Ultrasound-assisted extraction of phycobiliproteins from Spirulina (Arthrospira) platensis using protic ionic liquids as solvent

Algal Research 31 (2018) 454–462

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Ultrasound-assisted extraction of phycobiliproteins from Spirulina (Arthrospira) platensis using protic ionic liquids as solvent

T

Renata Débora Pinto Rodrigues, Felipe Carolino de Castro, Rílvia Saraiva de Santiago-Aguiar, ⁎ Maria Valderez Ponte Rocha Universidade Federal do Ceará, Technology Center, Chemical Engineering Department, Campus do Pici, Bloco 709, 60455–760 Fortaleza, Ceará, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Phycocyanin Allophycocyanin Phycoerythrin Protic ionic liquids Ultrasound

A new method that combines ultrasonic assisted extraction (UAE) with ionic liquids (ILs) was proposed to extract phycobiliproteins from the microalgae Spirulina (Arthrospira) platensis. Extraction of the pigments was carried out in an ultrasonic bath at 25 °C and at a frequency of 25 kHz. The effects of pH and solvent:biomass ratio were evaluated through a central rotational composite design and response surface methodology were used to determine the best extraction conditions. Solvents used were protic ionic liquids (PILs) 2-hydroxy ethylammonium acetate (2-HEAA), 2-hydroxy ethylammonium formate (2-HEAF), their equimolar mixture (2-HEAA+2-HEAF) (1:1 v/v) and the commercial ionic liquid, 1-butyl-3-methylimidazolium chloride [Bmim][Cl] and sodium phosphate buffer (0.1 M) as a control. Results showed that the PILs were able to extract the phycobiliproteins from microalgae. The pH was the most significant variable. Solvent:biomass ratio was also significant in the extraction process. The highest concentrations of phycobiliproteins were observed using 2-HEAA +2-HEAF as solvent at pH 6.50 and solvent:biomass ratio 7.93 mL·g−1 within 30 min of extraction. Allophycocyanin was the pigment extracted in greater quantity (6.34 mg·g−1), followed by phycocyanin (5.95 mg·g−1) and phycoerythrin (2.62 mg·g−1). Scanning electron microscopy (SEM) revealed that the ultrasound affect the cellular structure of the microalgae.

1. Introduction Microalgae are organisms capable of synthesizing and accumulating valuable chemical compounds, such as polysaccharides, unsaturated fatty acids and a range of pigments, among them phycobiliproteins [1]. Phycobiliproteins are proteins that capture light and act as accessory photosynthetic pigments in cyanobacteria and red algae. They are divided into three subgroups: phycocyanin, allophycocyanin and phycoerythrin, each with its own characteristic color, absorbing light in a specific region of the spectrum, 615 nm, 652 nm and 562 nm, respectively [2]. These pigments are commonly used in cosmetic [3], pharmaceutical [4,5] and food industries [6–8] by their color and interesting spectral and physiological properties, such as antioxidant [9,10], anti-inflammatory [11,12] and anticancer activity [13]. Have also been used as fluorescent probes in histochemistry, flow cytometry, microscopy and fluorescence immunoassays [14]. More recently, phycobiliproteins have called attention by their potential application in the energy field, namely in the production of “dye-sensitized solar cells” (DSSCs). DSSCs are non-tracking concentrators that redirect solar radiation into simple slab waveguides to be collected by a photovoltaic

⁎

cell mounted at the edge of the slab [15]. Some microalgae, such as Spirulina (Arthrospira) platensis, have a rigid cell wall that makes it difficult to extract specific components. To overcome this barrier, a cellular disruption operation is required in order to allow access to the internal components, facilitating the extraction process. This step is particularly important, because the contents of the extracted biomolecules are determined according to the disruption method and device used, and using an appropriate method and device is a key factor in increasing the biomolecule extraction efficiency. Therefore, many breaking techniques have been proposed: grinding, sonication, microwave, enzymatic treatment and high pressure cellular disruption to recover different components [16]. Ultrasound assisted extraction (UAE) has been used successfully in the extraction of various compounds and is easily implemented for large-scale industrial application [17,18]. The main phenomenon that occurs in exposure to ultrasound is cavitation. The cavitation is characterized by the violent collapse of bubbles in an alternating pressure field. Cavitation bubbles in the aqueous suspension of algae produce severe and localized short-term pressure increases as well as microstreaming effects (movement of liquid around gas bubbles formed by cavitation)

Corresponding author at: Chemical Engineering Department, Universidade Federal do Ceará, Campus do Pici, Bloco 709, Fortaleza, CE 60455-760, Brazil. E-mail address: [email protected] (M.V.P. Rocha).

https://doi.org/10.1016/j.algal.2018.02.021 Received 13 November 2017; Received in revised form 22 January 2018; Accepted 16 February 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.

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2.2. Synthesis and characterization of protic ionic liquids

and shock waves that promote the rupture of algae cells [19]. Some authors have reported the use of UAE in the extraction of phycobiliproteins from different species of microalgae [20–23]. In order to maximize the biotechnological and commercial potential of phycobiliproteins, new more efficient, economical and environmentally safe extraction methods must be developed. In this context, ionic liquids appear as an attractive and innovative alternative for the extraction of these biomolecules. Taking into account its wide versatility and capacity for reuse. ILs are salts, composed only of ions, with melting points below 100 °C, have low vapor pressure, high solvability and better chemical and thermal stability when compared to organic solvents. IL shave adjustable properties since their physical properties (polarity, hydrophobicity and viscosity) can be controlled by the exchange or combination of cations and anions. Due to their wide window of polarity, they are used in the most varied processes: from organic synthesis [24], extraction of metals [25,26], biocatalysis [27,28] to pretreatment of biomass [29,30]. Despite the many interesting features, most ILs have high cost disadvantages and low biodegradability. However, they have the advantage that they can be regenerated and reused [31]. ILs are classified into two groups: aprotic (AILs) and protic (PILs). AILs are mainly based on imidazolium and pyridine cations that are easier to use when compared to PILs. To get an idea, 250 g of the AIL used in this work, [Bmim][Cl], cost US$ 2823 [32] while 1 L of PIL 2-HEAF, considering only the value of the reagents, costs approximately US$ 9.28. PILs are synthesized by the transfer of protons from a Brønsted acid to a Brønsted base [33,34]. Among the advantages in the use of PILs we can mention: simplified synthesis, possibility of recycling and reuse and greater biodegradability in comparison to AILs [35]. In this context, a new method that combines UAE with protic ionic liquids has been proposed to obtain phycobiliproteins from Spirulina (Arthrospira)platensis. The performance of extraction using different protic ionic liquids was compared with sodium phosphate buffer and the aprotic ionic liquid ([Bmim][Cl]). The variables pH and solvent:biomass ratio were optimized in order to maximize the concentration of the pigments. The recovery and reuse of the ionic liquids were evaluated. As the extraction of phycobiliproteins using protic ionic liquid is a new methodology, the data on the reuse of ILs in the extraction of phycobiliproteins are scarce. In this study, initial recovery trials using ammonium sulfate and dialysis were performed. Fractional precipitation of two steps ammonium sulfate promoted the salting out of unwanted proteins while concentrating the proteins of interest [36]. Ammonium sulfate was chosen as the precipitating agent because it maintains protein integrity, precipitates readily the phycobiliproteins, helps to reduce the amount of sample to be handled, and it is highly soluble at low temperatures [37].

Protic ionic liquids 2-hydroxy ethylammonium acetate (2-HEAA) and 2-hydroxy ethylammonium formate (2-HEAF) were synthesized through an acid-base neutralization reaction as described by [30,38]. Acetic acid (≥99.85%), formic (88–91%), monoethanolamine (99%) and aprotic ionic liquid [Bmim][Cl] (≥98%) acids were purchased by Sigma-Aldrich and were not subjected to further purification processes. After the synthesis, the PILs were stored at 25 ± 2 °C in dark bottles to avoid degradation by light. The product was characterized by nuclear magnetic resonance analysis and physico-chemical methods: pH, density and viscosity. Density and viscosity were measured using a digital densimeter (ANTON PAAR, DSA 5000, AUSTRIA) and a digital U-tube oscillation viscometer (ANTON PAAR, SVM 3000, AUSTRIA) at 25 °C, respectively. The pH of the PILs was obtained by a pHmeter (Tecnal TEC-5, São Paulo, Brazil). 1H NMR spectra were obtained on a 600 MHz Agilent DD2 (USA) spectrometer equipped with a reverse-detection 5 mm internal diameter (H-F/15N-31P) one-probe and z-axis field gradient. One-dimensional 1H spectra were acquired with a time of acquisition of 1 s, gain of 26, acquisition of 16 transients in a spectral window of 16 ppm and 32 k number of points. Data was processed using the TopSpin 3.0™ program. 2.3. Extraction and determination phycobiliproteins S. platensis was immersed in different solvent (protic ionic liquids (PILs): 2-hydroxy ethylammonium acetate (2-HEAA) and 2-hydroxy ethylammonium formate (2-HEAF); their equimolar mixture (2HEAA+2-HEAF) (1:1 v/v); the commercial ionic liquid, 1-butyl-3-methylimidazolium chloride [Bmim][Cl] and sodium phosphate buffer (0.1 M) as a control) and the extraction of phycobiliproteins was assessed using an ultrasonic device (UNIQUE, USC-1450,BRAZIL) with a frequency of 25 kHz and 25 ± 2 °C, for 30 min. A DCCR 22 experimental design was used to evaluate of the effect of dependent variables pH and solvent-to-biomass ratio in the obtained concentrations of phycobiliproteins. The levels and ranges adopted in the planning are summarized in Table 1. The design used 4 factorial points, 4 axial points and 3 repetitions at the central point, resulting in 11 assays by solvent. The pH was chosen as a variable in the study because it is a critical factor in the extraction processes and it directly affects the solubility of biomolecules [1]. In addition to interfering with solubility, pH affects the kinetic constants of proteins. High pH values alter the net charge of proteins, causing electrostatic repulsion and disruption of some hydrogen bonds, destabilizing them [1]. Silveira et al. [39], on the other hand, optimizing the process of extraction of the phycocyanin of S. platensis by the conventional method with different solvents, showed that the solvent:biomass ratio strongly influenced the pigment extraction process and that the maximum value was obtained using the lowest solvent:biomass ratio. After extraction, 0.5 mL aliquots were withdrawn from the reaction medium and centrifuged at 6000 rpm for 10 min (GMCLAB GILSON, CAPSULEFUGE PMC-880, JAPAN). The optical density of the supernatants was determined by spectrophotometry (BIOCHROM, LIBRA S11, UK) at wavelengths 562 nm, 615 nm and 652 nm. The concentrations of phycocyanin, allophycocyanin and phycoerythrin were

2. Material and methods 2.1. Microalgae and cultivation conditions The Cyanophyte Spirulina (Arthrospira) platensis was obtained from an external monoculture carried out in a recirculation system in polyethylene boxes with a volume of 500 L at 30 ± 2 °C for 20 days. The culture was enriched with urea [CH4N2O] (0.1 g·L−1) and triple superphosphate [Ca(H2PO4)2H2O] (0.01 g·L−1) (SYNTH) with aquaculture effluents to replace losses by evaporation. Alkalinity of the selective medium was controlled by addition of 10 g·L−1 sodium bicarbonate [NaHCO3]. The optical density of the culture medium was monitored at 680 nm using a spectrophotometer (HACH, DR2700, PORTUGAL) and the biomass of S. platensis was filtered daily through a blanket of synthetic glass fiber with 60 μm of pore size. The biomass was then dried in oven with air recirculation at 60 °C for 24 h and ground until a fine powder was obtained and particles of 0.25 mm to 0.84 mm diameter were selected. The biomass powder was stored in capped flasks at 25 °C until studies were carried out.

Table 1 Variables and levels for central rotational compound design 22 (DCCR) using ultrasound in the extraction of phycobiliproteins from Spirulina (Arthrospira) platensis. Variables

pH Solvent:biomass ratio

455

Unit

– (mL·g−1)

Levels –α

–1

0

+1

+α

4.38 7.93

5.00 10.00

6.50 15.00

8.00 20.00

8.62 22.07

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0.9

estimated by Eqs. 1, 2 and 3, respectively [40].

OD615 − 0.474 × OD652 5.34

CAP (g . L−1) =

OD652 − 0.208 × OD615 5.09

CPE (g . L−1) =

OD562 − 2.41 × CP − 0.849 × CAP 9.62

0.8

(1)

0.7

-1 Phycocyanin (g.L )

CP (g . L−1) =

(2) (3)

where CP is the phycocyanin concentration (g·L−1); CAP is allophycocyanin concentration (g·L−1) and CPE is phycoerythrin concentration (g·L−1); OD562, OD615 and OD652 the optical densities of the samples at 562 nm, 615 nm and 652 nm, respectively.

a

a

a

A

a

0.6 0.5

b

0.4 0.3 0.2 0.1 0.0

2.4. Data analysis Data analysis was performed using ANOVA and the Tukey test to identify significant differences between means using Statistica® 10.0 software. The significance level considered for the difference between the means was 95% (p < 0.05). All analyzes were performed in triplicate and the results presented as mean ± standard deviation.

er Buff

F AF -HEAA l] HEA 2 2-HE m][C + 2A Bmi A [ E H 2

0.9

a

Allophycocyanin (g.L-1)

0.8

2.5. Scanning electron microscopy (SEM) The changes in the cellular structure of the microalgae were observed by scanning electron microscopy (SEM - HITACHI TM 3000 TABLETOP MICROSCOPE), under the best extraction conditions of extraction, one control (whole biomass) was used for comparison purposes. To prepare the samples, they were dispersed in distilled water, followed by pipetting on glass slides and drying at room temperature (25 °C/24 h).

0.7 0.6

At the end of the ultrasonic extraction process (25 kHz; ratio S:B 7.93 mL·g−1; 30 min) using 2-HEAA+2-HEAF (pH 6.50), the sample was filtered for the separation of the biomass and the supernatant submitted to consecutive precipitations with ammonium sulfate (25% and 45% w/v saturation) and centrifuged (6500 rpm/4 °C/15 min). Solid ammonium sulfate was added to obtain 25% (w/v) saturation, remaining at 4 °C for 4 h and then centrifuged at 6500 rpm for 15 min at 4 °C (EXCELSA 280 R, BRAZIL). The precipitate obtained was discarded and the supernatant subsequently added with ammonium sulfate until reaching 45% (w/v) saturation. The sample was refrigerated (4 °C) overnight, then it was centrifuged again. Approximately 3 mL of the precipitate obtained from ammonium sulfate precipitation was resuspended in 0.1 M sodium phosphate buffer (pH 7.00) and dialysed on the Sephadex G-25 M (8 × 1.6 × 5 cm) PD-10 column (GE HEALTHCARE). Supernatant, containing IL with remaining proteins, was reused in the extraction process and resubmitted to new precipitations with ammonium sulfate as previously mentioned. The operational stability of IL was evaluated in 3 different cycles. The phycobiliproteins were quantified according to the protocol described in item 2.3.

a

B

b

0.5 0.4 0.3

c

0.2 0.1 0.0

2.6. Recovery and reuse of ionic liquid

a

er Buff

AF 2-HE

l] AA EAF mim][C 2-HE +2-H [B A A 2-HE

0.9

C

Phycoerythrin (g.L-1)

0.8 0.7 0.6 0.5 0.4 0.3 0.2

a b

er Buff

a c

0.1 0.0

a

AF 2-HE

l] AA EAF m][C 2-HE +2-H Bmi [ A A 2-HE

Fig. 1. Influence of the solvent on the maximum concentrations of phycocyanin (A), allophycocyanin (B) and phycoerythrin (C) using ultrasound under the following conditions: pH 8.0 and solvent:biomass ratio 10 mL·g−1 for the buffer; pH 6.5 and solvent:biomass ratio7.93 mL·g−1 for protic ionic liquids and pH 4.38 and solvent:biomass ratio 15 mL·g−1 for [Bmim][Cl] (1-butyl-3-methylimidazolium chloride). Different lower case letters in the same column indicate significant differences as determined by Tukey's test (p < 0.05). Data shown is the mean ± SD, n = 3. Description: In Fig. 1 it is possible to compare the performance of all solvents evaluated under the best extraction conditions for each pigment (phycocyanin, allophycocyanin and phycoerythrin).

3. Results and discussion 3.1. Synthesis and characterization of ionic liquids The structures of the synthesized protic ionic liquids were confirmed by nuclear magnetic resonance (1H NMR) (Fig. 1 ESI and Fig. 2 ESI – Supplementary material). Observing the spectrum of 2-HEAA, there was a considerable change in the chemical shift of the amine group hydrogen from monoethanolamine (from 5.12 ppm to 6.28 ppm) and a small variation of the carboxylic acid carbon. Indicating that IL formation occurs through non-covalent intermolecular interactions of the amine group of monoethanolamine with the carboxylic acid oxygen. 456

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Fig. 2. Scanning Electron Microscopy (SEM) of Spirulina (Arthrospira) platensis samples submitted to ultrasound. Under the best extraction conditions: (A) control 1000× increased; (A') control increased 2000×; (B) ultrasound 1000× increased; (B′) ultrasound 2000× increased. Description: in Fig. 2 it is possible to observe through SEM the Spirulina (Arthrospira) platensis cell before and after the extraction of the pigments by ultrasound.

and Saba et al. [48] reported density around 1.08 g·cm−3 at 25 °C for this IL.

The chemical displacements of the other hydrogens did not vary, since the nature of the intermolecular interaction is not strong enough to cause large variations [41]. In the spectrum of 2-HEAF the broader peaks refer to the moving proton of the structure corresponding to the NH3+ function (7 ppm) and to the hydroxyl peak belonging to the cation (3.65 ppm). The peak located at 4.27 ppm refers to the ethyl (CH2) group attached to the carboxyl group of the anion. The peaks at 3.52 ppm and 2.86 ppm correspond, respectively, to the ethyl and methyl (CH3) groups attached to the cation. The spectra obtained were in agreement with those reported by [30,41,42]. In Table 2 are presented the physico-chemical properties of the solvents used in this research. Among the PILs, 2-HEAA presented higher viscosity and pH and the highest density was found for 2-HEAF. The 2-HEAA+2-HEAF presented intermediate values for all parameters evaluated. The mixture of ILs with other ILs aims to improve their target properties while maintaining favorable characteristics. Some properties of such mixtures can be described by the behavior of simple mixtures between two pure ILs [43]. Similarly, Annat et al. [43] when mixing [P6,6,6,14][NTf2] and [C3mpyr][NTf2] observed that the physical properties of this system deviated significantly from the behavior of individual ILs. The values of density and viscosity of the synthetized PILs consistent with presented by [41,44–46]. Huddleston et al. [47]

3.2. Extraction of phycobiliproteins Some microalgae are provided with a rigid cell wall which prevents or hinders the extraction of specific components. To overcome this barrier, an initial cellular disruption operation is necessary in order to allow access to the internal components, facilitating the extraction process [49]. The results presented in Table 3 showed that the ultrasound combination with ILs was efficient in the extraction of phycobiliproteins from microalgae. The buffer was more effective at pH 8.0 and solvent:biomass ratio 10 mL·g−1 (CP, CAP, CPE of 0.80; 0.54; 0.15 g·L−1, respectively) and [Bmim][Cl] at acid pH (4.38) and intermediate solvent:biomass ratio (15 mL·g−1) (CP, CAP, CPE of 0.36; 0.18; 0.09 g·L−1, respectively). A small variation was observed in the performance of PILs at the extraction of allophycocyanin comparing to phycocyanin. In turn, [Bmim][Cl] and sodium phosphate buffer extracted much less allophycocyanin than phycocyanin. This result demonstrates the greater diffusional power and the greater interaction of the PILs with the pigments. Phycoerythrin was the least extracted pigment for all solvents used, what was already expected, since in bluegreen microalgae, such as S. platensis, this pigment is naturally present in smaller proportions, being more abundant in red algae [42]. All PILs extracted better under assay conditions 7: pH 6.5 and ratio S:B 7.93 mL·g−1. The best results with 2-HEAF were 0.73; 0.77; 0.31 g·L−1 to CP, CAP and CPE, respectively. For 2-HEAA were 0.68; 0.74; 0.30 g·L−1 to CP, CAP and CPE, respectively, and for 2-HEAA+2-HEAF were 0.75; 0.80; 0.33 g·L−1 to CP, CAP and CPE, respectively. In Fig. 1 it is possible to compare the performance of the solvents in obtaining phycocyanin, allophycocyanin and phycoerythrin. It can be seen that statistically there was no significant difference (p < 0.05) between PILs and buffer performance in obtaining phycocyanin, although the highest concentration was obtained with the mixture 2-HEAA+2-HEAF

Table 2 Physico-chemical characterization of the solvents used in the extraction process measurements performed at 25 °C. Solvent

Density (g·cm−3)

Viscosity (mPa·s)

pH

2-HEAF 2-HEAA 2-HEAF+ 2-HEAA (1:1) Sodium phosphate buffer 0.1 M

1.1936 1.1510 1.1694 1.0202

81.8 2696.9 229.9 0.89

7.73 9.71 8.84 7.00

2-HEAF: 2-hydroxy ethylammonium acetate, 2-HEAA: 2-hydroxy ethylammonium formate, 2-HEAA+2-HEAF: 2-hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate (1:1).

457

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Table 3 Observed versus predicted values of phycocyanin, allophycocyanin and phycoerythrin concentrations (g·L−1) with different solvents using ultrasonic assisted extraction for 30 min. Data shown is the mean ± SD, n = 3. Phycocyanin (g·L−1) Assay

1 2 3 4 5 6 7 8 9 10 11

pH

5 5 8 8 4.38 8.62 6.5 6.5 6.5 6.5 6.5

S:B ratio (mL·g−1)

10 20 10 20 15 15 7.93 22.07 15 15 15

Allophycocyanin (g·L−1) Assay pH S:B ratio (mL·g−1) 1 2 3 4 5 6 7 8 9 10 11

5 5 8 8 4.38 8.62 6.5 6.5 6.5 6.5 6.5

Phycoerythrin (g·L−1) Assay pH 1 2 3 4 5 6 7 8 9 10 11

5 5 8 8 4.38 8.6 6.5 6.5 6.5 6.5 6.5

10 20 10 20 15 15 7.93 22.07 15 15 15 S:B ratio (mL·g−1) 10 20 10 20 15 15 7.93 22.07 15 15 15

Buffer

2-HEAF

2-HEAA

2-HEAA+2-HEAF

[Bmim][Cl]

Obs

Pred

Obs

Pred

Obs

Pred

Obs

Pred

Obs

Pred

0.37 0.17 0.80 0.43 0.24 0.43 0.60 0.27 0.41 0.47 0.50

0.38 0.21 0.71 0.36 0.19 0.53 0.65 0.28 0.46 0.46 0.46

0.30 0.16 0.67 0.25 0.06 0.31 0.73 0.27 0.38 0.37 0.34

0.33 0.16 0.67 0.22 0.05 0.33 0.72 0.29 0.36 0.36 0.36

0.20 0.12 0.30 0.24 0.03 0.47 0.68 0.28 0.33 0.32 0.35

0.28 0.09 0.48 0.31 0.03 0.32 0.53 0.28 0.33 0.33 0.33

0.30 0.12 0.69 0.31 0.03 0.57 0.75 0.36 0.53 0.52 0.51

0.31 0.13 0.74 0.37 0.03 0.50 0.70 0.32 0.52 0.52 0.52

0.27 0.25 0.16 0.15 0.36 0.09 0.23 0.16 0.26 0.22 0.29

0.30 0.26 0.14 0.12 0.33 0.12 0.21 0.17 0.26 0.26 0.26

Buffer Obs 0.21 0.07 0.54 0.20 0.10 0.24 0.27 0.07 0.18 0.22 0.27

Pred 0.18 0.08 0.44 0.15 0.10 0.33 0.35 0.07 0.22 0.22 0.22

2-HEAF Obs 0.41 0.19 0.64 0.19 0.07 0.25 0.77 0.21 0.36 0.36 0.30

Pred 0.40 0.16 0.63 0.16 0.10 0.26 0.75 0.24 0.34 0.34 0.34

2-HEAA Obs 0.10 0.04 0.42 0.15 0.02 0.74 0.74 0.31 0.42 0.44 0.44

Pred 0.21 0.07 0.68 0.32 0.02 0.50 0.57 0.23 0.43 0.43 0.43

2-HEAA+ 2-HEAF Obs Pred 0.35 0.35 0.06 0.079 0.82 0.86 0.32 0.39 0.02 0.02 0.67 0.61 0.80 0.78 0.31 0.26 0.54 0.56 0.54 0.56 0.59 0.56

[Bmim][Cl] Obs 0.17 0.11 0.08 0.03 0.18 0.07 0.05 0.05 0.10 0.10 0.11

Pred 0.15 0.11 0.06 0.04 0.18 0.07 0.08 0.03 0.10 0.10 0.10

Buffer Obs 0.06 0.02 0.15 0.04 0.03 0.06 0.06 0.01 0.04 0.04 0.05

Pred 0.05 0.02 0.12 0.03 0.03 0.09 0.09 0.01 0.04 0.04 0.05

2-HEAF Obs 0.17 0.09 0.27 0.08 0.03 0.10 0.31 0.07 0.12 0.11 0.12

Pred 0.16 0.07 0.27 0.06 0.05 0.11 0.30 0.09 0.12 0.12 0.12

2-HEAA Obs 0.05 0.03 0.19 0.07 0.01 0.26 0.30 0.13 0.20 0.22 0.21

Pred 0.09 0.04 0.28 0.12 0.01 0.18 0.26 0.10 0.21 0.21 0.21

2-HEAA+2-HEAF Obs Pred 0.12 0.12 0.03 0.04 0.34 0.37 0.10 0.14 0.01 0.01 0.30 0.26 0.33 0.30 0.13 0.09 0.21 0.23 0.23 0.23 0.26 0.23

[Bmim][Cl] Obs 0.052 0.04 0.03 0.02 0.09 0.05 0.04 0.03 0.06 0.06 0.06

Pred 0.06 0.05 0.03 0.03 0.08 0.05 0.03 0.02 0.06 0.06 0.06

(⁎)In bold, the highest concentration values observed. S:B Ratio: solvent:biomass ratio, Buffer: sodium phosphate buffer, 2-HEAF: 2-hydroxy ethylammonium acetate, 2-HEAA: 2-hydroxy ethylammonium formate, 2-HEAA+2-HEAF: 2hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate, [Bmim][Cl]: 1-butyl-3-methylimidazolium chloride.

(0.75 ± 0.01 g·L−1). The [Bmim][Cl] was the least efficient solvent, having extracted 0.36 ± 0.02 g·L−1 of phycocyanin. The macrostructure of phycobilissomes, which contains phycobiliproteins, consists of a nucleus (composed of allophycocyanin) surrounded by phycocyanin and phycoerythrin organized at the periphery of the structure in the form of rods [50]. This explains because the buffer has been able to extract substantially the same amounts of phycocyanin as the PILs. Phycocyanin is more readily available and therefore easier to extract than allophycocyanin which is located inside the structure. In addition, according to SEM, ultrasound interfered very little in the structure of the microalgae cell, which may have hindered the release of allophycocyanin in the solvents (Fig. 2). Observing the performance of the solvents in the extraction of allophycocyanin and phycoerythrin, it can order them, from highest to lowest concentration, as follows: PILs > buffer > [Bmim][Cl]. The highest concentrations of allophycocyanin (0.80 ± 0.01 g·L−1) and phycoerythrin (0.33 ± 0.01 g·L−1) were achieved using 2-HEAA+2-HEAF, although there was no significant difference (p < 0.05) between this solvent and the remaining PILs. For Elgharbawya et al. [29], cation and anion are equally important for the performance of ionic liquids. The basicity of the anion correlates with

its ability to form hydrogen bonds. The higher the anion chain, the lower its ability to form hydrogen bonds and the lower the solubility of the target molecule in IL. The structure and size of the cation determine the melting point and the polarizability of the ILs, directly influencing the solubilization of the proteins. Araki et al. [51] have observed that the insertion of hydroxyl, ether or amide groups to ILs increases their affinity for proteins. This helps to explain why 2-HEAA+2-HEAF improves performance against 2-HEAA and 2-HEAF isolated. The low efficiency of the [Bmim][Cl] must be linked to its high hydrophobicity associated with the flatness of the ring of the imidazolium cation (which promotes a less steric effect) that appear to be important for the increase of the toxicity of the ionic liquids. These parameters favor the interactions of the ionic liquids with the proteins, provoking their denaturation [39]. Preliminary experiments were conducted at 40 kHz for different times using sodium phosphate buffer (data not show) and it was observed that after 30 min occurred a decrease in the yield phycobiliproteins. Probably under more drastic sonication conditions, cavitation effects would promote extensive rupture of algae cells releasing undesirable compounds and can denature the extracted phycobiliproteins. 458

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were 0.40 g·L−1, 0.42 g·L−1 and 0.10 g·L−1 (data not show), lower concentrations than those obtained with ultrasound. These results indicate that the ultrasound promotes a rupture of the cell, increasing the extraction efficiency of the molecules of interest. The yields of phycocyanin obtained in this study were higher than those observed by Abalde et al. [20] 0.007 g·L−1 (distilled water, 4 °C, 5 min) and slightly lower than those achieved by Hadyanto et al. [17] 1.05 g·L−1 (ethanol, 200 W, 52.5 °C, 42 min), with a lower energy consumption than the latter. Under sonication conditions, cavitation effects would promote extensive rupture of algae cells releasing undesirable compounds [22,23]. The Pareto chart for the extraction of phycobiliproteins is presented in Fig. 3. The linear and positive effect of pH was the most relevant in the extraction of the pigments. By increasing the pH of the medium, the phycobiliproteins remain negatively charged which favors the interaction with the solvent, facilitating the extraction process [23]. The negative and linear effect of the solvent:biomass ratio indicates that the extraction will be more efficient using lower volumes of solvent. This effect may be related to the greater mass transfer between the solute and the solvent under these conditions [18]. However, in very low solvent volumes, the correct interaction with the solute does not occur [39]. The ANOVA for the phycobiliproteins extraction models is shown in Table 4. The models fitted well to the observed responses, which can be evidenced by the high R2and Fcalc > Ftab. The concentration values of the phycobiliproteins predicted by the models showed agreement with the results obtained experimentally. The models for the extraction of phycocyanin, allophycocyanin and phycoerythrin with 2-HEAA+2HEAF on ultrasound are represented by Eqs. 4, 5 and 6.

PHYC O C YANIN

A (1)pH(L)

52.703

-43.1552

(2)Ratio s:b(L)

pH(Q )

-33.9832

1Lby2L

Ratio s:b(Q )

-10.9413

-1.87878

p=.05 Estimating the standardiz e d e ffe ct (absolute value )

ALLO PHYC O C YANIN

B (1)pH(L)

(2)Ratio s:b(L)

pH(Q )

1Lby2L

Ratio s:b(Q )

-9.90337

-3.56048

-1.44738

Cphycocyanin(g·L − 1) = −0.26pH2 − 0.10pH × Ratio S : B + 0.33pH − 0.27Ratio S: B + 0.52

(4)

p=.05 Estimating the standardiz e d e ffe ct (absolute value )

Table 4 ANOVA for the extraction model of phycocyanin, allophycocyanin and phycoerythrin on ultrasound using 2-HEAA +2-HEAF (2-hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate) as solvent.

PHYCOERYTHRIN

C (1)pH(L)

Quadratic sum (QS)

Degrees of freedom (DL)

Quadratic mean (QM)

a

Phycocyanin Regression Residue Lack of adjustment Pure error Total R2 b Ftab

0.4757 0.0147 0.4904 0.0145 0.4904 0.97 a F(5.5) = 5.05

5 5 10 3 10

0.0951 0.0029 0.0490 0.0048 0.0490

32.40

Allophycocyanin Regression Residue Lack of adjustment Pure error Total R2 b Ftab

0.7115 0.01524 0.7267 0.0135 0.7267 0.98 a F(5.5) = 5.05

5 5 10 3 10

0.1423 0.0030 0.0727 0.0045 0.0727

46.70

0.1249 0.0063 0.1313 0.0050 0.1313 0.95 a F(5.5) = 5.05

5 5 10 3 10

0.0250 0.0013 0.0131 0.0017 0.0131

19.71

Source of variation (2)Ratio s:b(L)

-8.21214

pH(Q )

1Lby2L

Ratio s:b(Q )

-4.49589

-2.80195

-1.63553

p=.05 Estimating the standardized effect (absolute value)

Fig. 3. Pareto chart of effects estimated for the variables (pH and solvent:biomass ratio) extraction of phycocyanin (A), allophycocyanin (B) and phycoerythrin (C) on ultrasound using 2-HEAA+2-HEAF (2-hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate) as solvent. (1) pH (L): linear pH effect; (2) Ratio S:B (L): linear effect of the solvent:biomass ratio; pH (Q): quadratic pH effect; 1Lby2L: interaction between the linear effect of pH and the linear effect of the solvent:biomass ratio; Ratio S:B (Q): quadratic effect of the solvent:biomass ratio. Description: Fig. 3 shows the Pareto diagram of the variables studied (pH and solvent:biomass ratio) in the extraction of phycobiliproteins using 2-HEAA+2-HEAF as solvent.

Phycoerythrin Regression Residue Lack of adjustment Pure error Total R2 b Ftab 95% de confiança

For this reason the experiments were not carried out at a higher frequency. Also, experiments were performed without ultrasound under conditions of 25 °C (ultrasound operating temperature) for 30 min and the concentrations of phycocyanin, allophycocyanin and phycoerythrin

a b

459

Fcalculated = QMregression/QMresidue,where QM = quadratic mean. Ftabulated.

Fcalc

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R.D.P. Rodrigues et al.

800

-1 )

Phycocyanin (mg.L

600 400 200 0 0 -20 0 -40 24

22

9.

20

0.6 0.5 0.4 0.3 0.2

0

5 8.

5 7.

0.1

5 6.

0 4.

6

5 4.

-1 )

g

8

0.0

pH

5 5.

10

0 5.

L. (m

12

0 6.

B S:

14

0.7

0 7.

16

0 8.

18

tio Ra

B

-1

0 100

phycocyanin allophycocyanin phycoerythrin

0.8

Phycobiliproteins (g.L )

A

0.9

> 800 < 700 < 500 < 300 < 100 < -100

L.g Allophycocyanin (m -1 )

800 600

2° cycle

3° cycle

Fig. 5. Recycle 2-HEAA+2-HEAF (2-hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate). Extractions performed on ultrasound (25 kHz, solvent:biomass ratio 7.93 mL·g−1, pH 6.50, 30 min). Data shown is the mean ± SD, n = 3. Description: Fig. 5 shows the cycles of reuse of ionic liquid 2-HEAA+2-HEAF in the extraction of phycobiliproteins.

> 1000 < 900 < 700 < 500 < 300 < 100 < -100 < -300

0 120 0 100

1° cycle

Cphycoerythrin(g·L − 1) = −0.10pH 2 + 0.17pH − 0.15Ratio S: B + 0.23 (6)

Fig. 4. Response surface for extraction of phycocyanin (A), allophycocyanin (B) and phycoerythrin (C) on ultrasound as a function of pH by the solvent:biomass ratio using 2HEAA+2-HEAF (2-hydroxy ethylammonium acetate +2-hydroxy ethylammonium formate) as solvent. Description: Fig. 4 shows the response surfaces as a function of pH by the solvent: biomass ratio in the extraction of phycobiliproteins using 2-HEAA+2-HEAF as solvent.

where: ratio S:B the amount of solvent (mL) from the biomass (g). Analyzing the response surfaces for phycobiliproteins extraction, it was observed that the optimum region was not reached but could be achieved by further reducing the solvent:biomass ratio and increasing the solvent pH (Fig. 4). This is not biochemically feasible because, as explained earlier, phycobiliproteins are unstable at elevated pH. In addition, at low solvent volumes there is no correct interaction between solute and solvent. PILs proved to be effective solvents in the extraction of phycobiliproteins of S. platensis, and one of the main advantages is the possibility of recovery and recycling of the solvents. After extraction to remove phycobiliproteins, 2-HEAA+2-HEAF was precipitated with ammonium sulfate in order to be used in a new extraction. The same IL can be reused in 3 different extraction cycles (Fig. 5). Phycocyanin seems to have been more affected in the process. The allophycocyanin concentration ranged from 0.80 g·L−1 ± 0.03 (1° cycle) to 0.51 ± 0.02 (3° cycle) and phycoerythrin concentration did not change (0.33 g·L−1). During the recycle of protic ionic liquid, there was a decrease in the yields of phycocyanin and allophycocyanin probably due to the presence of ammonium sulfate that was not completely removed in the dialysis. Besides that, ammonium sulfate present in the recovered IL favored the precipitation of these pigments, and thus not be quantified in the supernatant. This fact did not interfere in the extraction of phycoerythrin, because this phycobiliprotein only precipitates at high concentrations of ammonium sulfate (approximately 80% w/v) as cited by Su and Chen [23]. Also, the accumulation of other components during the extraction and recovery stages of protic ionic liquid may have negatively influenced its efficiency in the extraction of phycobiliproteins. For example, Reis et al. [30] evaluated the pretreatment of lignocellulosic material using the protic ionic liquid 2-HEAA, and the authors revealed that the accumulation of components in the recovered and reused protic ionic liquid (2-HEAA) reduced the efficiency of the lignin removal and yield of glucose obtained in the enzymatic hydrolysis from cashew apple bagasse. Against the foregoing, further studies should be performed to improve the extraction and recovery of liquids, ensuring a product to be obtained with appropriate purity.

Callophycocyanin (g·L − 1) = −0.24pH2 + 0.41 × pH − 0.37x Ratio S

4. Conclusions

400 2 00 0 0 -20 0 -40 -60

0

24

0

8.

0

0 6.

pH

0 5.

6

0 4.

C

8

5 4.

-1 )

g L.

10

5 5.

(m

12

5 6.

B S:

14

0 7.

16

5 7.

tio Ra

18

5 8.

20

9.

22

> 300 < 220 < 120 < 20 < -80 < -180

500 400

Phycoeryhtrin (mg

300 200 100 0 0 -10

.L-1)

0 -2 0 0 -3 0 24

0 8.

0 6.

0 5.

8

pH

0 4.

6

5 4.

-1 )

g L.

10

5 5.

(m

12

5 6.

B S:

14

0 7.

16

5 7.

tio Ra

18

5 8.

20

0 9.

22

: B + 0.56

(5)

In this work an alternative methodology for the extraction of 460

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phycobiliproteins using ultrasound and ionic liquids was proposed and optimized. The synthesized protic ionic liquids proved to be more efficient than the sodium phosphate buffer and the commercial ionic liquid [Bmim][Cl] in the extraction of the phycocyanin, allophycocyanin and phycoerythrin. Allophycocyanin was the pigment extracted in greater quantity, followed by phycocyanin and phycoerythrin. Under the conditions of operation of the study, the best yields were obtained using 2-HEAA+2-HEAF as solvent at the frequency of 25 kHz, pH 6.50, solvent:biomass ratio of 7.93 mg·L−1 with 30 min of extraction.

[12]

[13] [14] [15]

Author contributions statement

[16]

Renata Débora Pinto Rodrigues contributed to conception of the experiments performing experiments, analysis and interpretation of the data, and writing the manuscript. Felipe Carolino de Castro contributed to conception of the experiments, performing experiments. Rílvia Saraiva de Santiago-Aguiar contributed to analysis and interpretation of the data and revising the manuscript. Maria Valderez Ponte Rocha contributed to analysis and interpretation of the data and revising the manuscript. Funding information was provided in Acknowledgements.

[17]

[18]

[19] [20]

Acknowledgements [21]

This research was financial supported by Brazilian research funding agency FUNCAP (Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

[22]

[23]

Conflict of interest statement

[24] [25]

No conflicts, informed consent, human or animal rights applicable in this work.

[26]

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2018.02.021.

[27]

References

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