Effective Astaxanthin Extraction from Wet Haematococcus pluvialis

2 days ago - (14, 15) SHS have been widely used as extractants in various applications such as the extraction of biodiesel and phenols and the recycli...
5 downloads 13 Views 2MB Size
Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Letter

Effective astaxanthin extraction from wet Haematococcus pluvialis using switchable hydrophilicity solvents Wen-Can Huang, Hui Liu, Weiwei Sun, Changhu Xue, and Xiangzhao Mao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04624 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Effective astaxanthin extraction from wet Haematococcus pluvialis using switchable hydrophilicity solvents

Wen-Can Huanga, Hui Liua, Weiwei Suna, Changhu Xuea,b, Xiangzhao Maoa,b*

a

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

b

Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266200, China

*

Corresponding author: Professor Xiangzhao Mao

Address: College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China Tel.: +86-532-82032660 Fax: +86-532-82032272 E-mail: [email protected]

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A novel approach based on switchable hydrophilicity solvents (SHS) was developed for extracting astaxanthin from wet microalgae. Dimethylaminocyclohexane (DMCHA) was used to extract astaxanthin from wet Haematococcus pluvialis, and the extraction efficiency reached 87.2%. Astaxanthin was recovered from the DMCHA without distillation by simply adding H2O and CO2.

KEYWORDS: Astaxanthin, Switchable hydrophilicity solvents, Dimethylaminocyclohexane, Microalgae, Haematococcus pluvialis

Introduction Astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione), a red-orange ketocarotenoid,1,2 has attracted attention due to its powerful antioxidant activity.1,3-5 Because of this activity, astaxanthin is widely used in food, pharmaceutical and cosmetic applications.1,6,7 Additionally, astaxanthin is commonly used as a pigment in the aquaculture and poultry industries because of its bright red color.4 Astaxanthin is derived from natural resources or prepared synthetically.5 The use of synthetic astaxanthin, which is produced from petrochemical sources, raises issues related to food safety and environmental pollution.5,8 Thus, natural astaxanthin is preferred over its synthetic form. Natural sources for astaxanthin include organisms, such as microalgae and yeast, and crustacean byproducts.8,9 Among the various natural

ACS Paragon Plus Environment

Page 2 of 20

Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

sources, Haematococcus pluvialis is considered to be the primary source for natural astaxanthin because it contains the highest concentration of astaxanthin.3,8,10 To produce astaxanthin from microalgae, three major steps are involved, including cultivation, harvest, and extraction.5 Of these three steps, astaxanthin extraction is considered to be the major challenge because the stress conditions that induce the accumulation of astaxanthin in microalgae also induce the formation of rigid and thick cell walls.4,10 Thick cell walls increase the mechanical and chemical resistance of microalgal cells

1

and inhibit solvent access to the cells.10,11 Cell

disruption is used to increase the extraction efficiency10; however, it also increases the energy that is consumed during processing.10 Moreover, because of the basic chemistry concept of “like dissolving like”, organic solvents such as dichloromethane, chloroform/methanol and hexane/ethanol are commonly used to extract astaxanthin from H. pluvialis. Conventional solvent extraction is easy to scale up; however, the main problem in using organic solvents is the high energy costs that are associated with separating astaxanthin from the solvent via distillation and evaporation.12 Moreover, the presence of moisture seriously hampers astaxanthin extraction by limiting the contact between the hydrophobic solvents and the wet microalgal cells.13 Thus, the recovery process of astaxanthin requires drying steps, and similar to cell disruption and distillation, drying the microalgae is also an energy consuming step. Therefore, an energy-efficient and direct method for extracting astaxanthin from wet microalgae without a biomass pretreatment or solvent evaporation would be an attractive approach. Until now, many methods, including the use of solvent, ultrasonication, acid, base and supercritical CO2, have been reported to enhance the extraction efficiency of astaxanthin from Haematococcus pluvialis.11 Recently, ionic

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

liquids were used to permeabilise the Haematococcus pluvialis at mild temperatures, and more than 70% of astaxanthin was extracted.1 In this study, a novel approach was designed for extracting astaxanthin from wet H. pluvialis based on switchable hydrophilicity solvents (SHS). Switchable hydrophilicity solvents are solvents that can be reversibly switched between a nonionic liquid, which is immiscible with water, and an ionic liquid, which is miscible with water, after a simple change in the system.14,15 SHS have been widely used as extractants in various applications such as the extraction of biodiesel and phenols and the recycling of multilayer packaging materials.12,16-19 In this study, for the first time, SHS are used to extract astaxanthin from wet H. pluvialis. An SHS-based extraction system successfully separated astaxanthin from wet H. pluvialis without any energyconsuming pretreatment processes, such as cell disruption or drying. Moreover, this approach does not have the energy costs associated with evaporating the solvents for recycling. Dimethylaminocyclohexane

(DMCHA), a switchable hydrophilicity

solvent, was chosen for the astaxanthin extraction from wet H. pluvialis in this study because of its low volatility and low water solubility, which are good properties for extraction and minimizing environmental concerns.

EXPERIMENTAL SECTION A schematic depiction of astaxanthin extraction from microalgae using SHS is shown in Figure 1. To extract astaxanthin from microalgae, 10 mL of DMCHA was added to various amounts (0.25, 5, and 12.5 g) of wet H. pluvialis with 80% water content. Next, microalgaeDMCHA mixtures were magnetically stirred for 1 h, 3 h, 6 h, and 24 h at room temperature. Then, the residual biomass was separated from the liquid phase via centrifugation at 7000

ACS Paragon Plus Environment

Page 4 of 20

Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

rpm for 5 min. After the cell debris was removed, a volume of H2O equal to twice the volume of the DMCHA was added, and the mixture was subjected to CO2 bubbling using a gas dispersion tube for 15 min. After the lipid layer that contained the astaxanthin floated to the top of the solution, the extracted astaxanthin was transferred to a new glass tube, resuspended in dichloromethane and filtered. Next, each sample was analyzed by high-performance liquid chromatography (HPLC; Agilent 1260 Infinity, Agilent Technologies, USA) to determine the amount of astaxanthin. Reconversion of the hydrophilic form back to the hydrophobic form was achieved by heating at 70 °C for 1 h to remove the CO2. The amount of DMCHA lost in the lipid layer was determined using NMR spectroscopy according to previously described methods.12 The total amount of astaxanthin that was extracted from the microalgae was determined to evaluate the extraction efficiency of DMCHA.1 Microalgae were disrupted using an ultrasonic processor and then freeze-dried prior to extraction in a chloroform/methanol mixture (2:1, v/v). Then, the amount of extracted astaxanthin was determined by HPLC analysis. The surface morphologies of H. pluvialis cells before and after the 24 h extraction with DMCHA were observed using scanning electron microscopy (SEM; Hitachi S4800, Hitachi, Japan). The samples were immersed in a liquid nitrogen bath and freeze-dried. The samples were placed on a metal substrate using carbon tape and then sputter coated with a platinum film. SEM images were taken at an acceleration voltage of 10 kV.

RESULTS AND DISCUSSION After the addition of H2O and CO2, hydrophobic DMCHA is converted into a hydrogen

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

carbonate ammonium salt, which is hydrophilic (Figure S1). The miscibility change of DMCHA is because of the acid-base reaction between DMCHA and hydrated CO2, which results in a bicarbonate salt of protonated DMCHA (eq (1)). DMCHA + H2O + CO2 ⇌ DMCHAH+ + HCO3-

(1)

The astaxanthin extraction efficiency of DMCHA was determined for varying microalgae/DMCHA ratios and times. As shown in Figure 2, the extraction efficiency continued to increase with the longer extraction times, up to 24 h, for all of the tested microalgae/DMCHA ratios. The highest astaxanthin extraction rate appeared during the time period from 1 to 3 h in the treatment, and no significant release of astaxanthin was detected after 6 h. At microalgae/DMCHA ratios of 100 mg mL-1 and 5 mg mL-1, the maximum extraction yields were 71.1% and 87.2%, respectively, after 24 h of extraction with DMCHA. Even at the microalgae/DMCHA ratio of 250 mg mL-1, the extraction efficiency of astaxanthin reached 57.7% after 24 h of extraction with DMCHA. After extraction, reconversion of the hydrophilic form, [DMCHAH+][HCO3-], back to the hydrophobic form of DMCHA was achieved through heating at 70 °C for 1 h to remove the CO2. Since the water solubility of DMCHA is significant,12 to reduce DMCHA losses, water saturated with DMCHA should be recycled. The major drawback of using DMCHA in astaxanthin extraction is the toxicity.12 The presence of SHS in the extracts is one of the main impediments to its wide utilization in extraction. Thus, recovery is always the main concern in the application of SHS. Recently, DMCHA losses in water have been reduced by increasing the salinity of the solution.12 The SEM images of the untreated microalgal cells and DMCHA-treated cells showed significant differences in the cell surface morphologies. The surfaces of the untreated cells appeared to be intact, uniform, smooth, and regular and had no apparent disruptions. In ACS Paragon Plus Environment

Page 6 of 20

Page 7 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

contrast, the surfaces of the DMCHA-treated cells showed significant morphological changes, including rough, distorted and squashed cell surfaces. Furthermore, as shown in Figure 3, the disruption of the cells and the release of their intercellular components from the ruptures on the surface of the microalgal cells were clearly observed in the SEM images, whereas the control experiment without the DMCHA treatment did not show any noticeable cell disruption. This result clearly shows that the DMCHA can disorder the membrane structures and cause disruption of the microalgal cells. The cell wall of H. pluvialis is very rigid because it consists of two layers, the trilaminar sheath and secondary wall.20 The trilaminar sheath is composed of algaenan, which is a tough, non-hydrolysable polymer,11,20 and the secondary wall beneath the trilaminar sheath is mainly composed of mannose and cellulose.11,20 According to previous studies, DMCHA, an amine, can interact with structural lipids12 and disrupt microalgal cell walls,12 which are resistant to extraction with traditional solvents such as chloroform and methanol. The ability of DMCHA to disrupt the cell walls was confirmed by the SEM observations of the DMCHA-treated H. pluvialis. Microalgal cell walls were completely disrupted by DMCHA, which suggested that DMCHA can disrupt the cellular structures of microalgae, release cellular components and extract lipid contents.

Conclusions In this study, a switchable, hydrophilic solvent-based method was demonstrated to be an efficient approach for extracting astaxanthin from wet microalgae without using additional cell disruption reagents or energy-intensive equipment. DMCHA was shown to extract 87.2% of the astaxanthin contained in wet H. pluvialis. The SHS-based astaxanthin extraction method is preferable to the conventional method because it can separate astaxanthin from

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DMCHA simply by adding water and CO2. It does not require distillation, which is the most energy-intensive step in solvent extraction, and the solvent system is recycled through the removal of CO2. Moreover, SHS-based extraction avoids additional energy-intensive cell pretreatment processes, including cell disruption and drying. Although this method must be improved because of the hazardous properties of DMCHA with regard to humans and the environment, we believe that further research will overcome the adverse biological effects and make SHS extraction a suitable method for recovering astaxanthin from microalgae.

ASSOCIATED CONTENT Supporting Information Materials and experimental details for additional characterizations.

ACKNOWLEDGMENTS This work was supported by China Agriculture Research System-48 and Major Special Science and Technology Projects in Shandong Province (2016YYSP016).

REFERENCES (1)

Desai, R. K.; Streefland, M.; Wijffels, R. H.; Eppink, M. H., Novel astaxanthin

extraction from Haematococcus pluvialis using cell permeabilising ionic liquids. Green Chem. 2016, 18, 1261-1267. (2)

Cheng, J.; Li, K.; Yang, Z.; Zhou, J.; Cen, K., Enhancing the growth rate and

astaxanthin yield of Haematococcus pluvialis by nuclear irradiation and high

ACS Paragon Plus Environment

Page 8 of 20

Page 9 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

concentration of carbon dioxide stress. Bioresour. Technol. 2016, 204, 49-54. (3)

Guerin, M.; Huntley, M. E.; Olaizola, M., Haematococcus astaxanthin:

applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210-216. (4)

Praveenkumar, R.; Lee, K.; Lee, J.; Oh, Y.-K., Breaking dormancy: an energy-

efficient means of recovering astaxanthin from microalgae. Green Chem. 2015, 17, 1226-1234. (5)

Panis, G.; Carreon, J. R., Commercial astaxanthin production derived by green

alga Haematococcus pluvialis: A microalgae process model and a techno-economic assessment all through production line. Algal Res. 2016, 18, 175-190. (6)

Yu, X.; Niu, X.; Zhang, X.; Pei, G.; Liu, J.; Chen, L.; Zhang, W., Identification

and mechanism analysis of chemical modulators enhancing astaxanthin accumulation in Haematococcus pluvialis. Algal Res. 2015, 11, 284-293. (7)

Praveenkumar, R.; Gwak, R.; Kang, M.; Shim, T. S.; Cho, S.; Lee, J.; Oh, Y.-

K.; Lee, K.; Kim, B., Regenerative astaxanthin extraction from a single microalgal (Haematococcus pluvialis) cell using a gold nano-scalpel. ACS Appl. Mater. Inter. 2015, 7, 22702-22708. (8)

Li, J.; Zhu, D.; Niu, J.; Shen, S.; Wang, G., An economic assessment of

astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnol. Adv. 2011, 29, 568-574. (9)

Weesepoel, Y.; Gruppen, H.; de Bruijn, W.; Vincken, J.-P., Analysis of

Palmitoyl Apo-astaxanthinals, Apo-astaxanthinones, and their Epoxides by UHPLCPDA-ESI-MS. J. Agr. Food Chem. 2014, 62, 10254-10263. (10)

Cheng, X.; Riordon, J.; Nguyen, B.; Ooms, M. D.; Sinton, D., Hydrothermal

disruption of algae cells for astaxanthin extraction. Green Chem. 2017, 19, 106-111. (11)

Kim, D.-Y.; Vijayan, D.; Praveenkumar, R.; Han, J.-I.; Lee, K.; Park, J.-Y.;

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chang, W.-S.; Lee, J.-S.; Oh, Y.-K., Cell-wall disruption and lipid/astaxanthin extraction from microalgae: Chlorella and Haematococcus. Bioresour. Technol. 2016, 199, 300-310. (12)

Samorì, C.; Barreiro, D. L.; Vet, R.; Pezzolesi, L.; Brilman, D. W.; Galletti, P.;

Tagliavini, E., Effective lipid extraction from algae cultures using switchable solvents. Green Chem. 2013, 15, 353-356. (13)

Huang, W.-C.; Kim, J.-D., Cationic surfactant-based method for simultaneous

harvesting and cell disruption of a microalgal biomass. Bioresour. Technol. 2013, 149, 579-581. (14)

Jessop, P. G.; Heldebrant, D. J.; Li, X.; Eckert, C. A.; Liotta, C. L., Green

chemistry: Reversible nonpolar-to-polar solvent. Nature 2005, 436, 1102-1102. (15)

Jessop, P. G.; Phan, L.; Carrier, A.; Robinson, S.; Dürr, C. J.; Harjani, J. R., A

solvent having switchable hydrophilicity. Green Chem. 2010, 12, 809-814. (16)

Boyd, A. R.; Champagne, P.; McGinn, P. J.; MacDougall, K. M.; Melanson, J.

E.; Jessop, P. G., Switchable hydrophilicity solvents for lipid extraction from microalgae for biofuel production. Bioresour. Technol. 2012, 118, 628-632. (17)

Samorì, C.; Cespi, D.; Blair, P.; Galletti, P.; Malferrari, D.; Passarini, F.;

Vassura, I.; Tagliavini, E., Application of switchable hydrophilicity solvents for recycling multilayer packaging materials. Green Chem. 2017, 19, 1714-1720. (18)

Fu, D.; Farag, S.; Chaouki, J.; Jessop, P. G., Extraction of phenols from lignin

microwave-pyrolysis oil using a switchable hydrophilicity solvent. Bioresour. Technol. 2014, 154, 101-108. (19)

Soh, L.; Eckelman, M. J., Green solvents in biomass processing. ACS

Sustainable Chem. Eng. 2016, 4, 5821-5837. (20)

Lee, S. Y.; Cho, J. M.; Chang, Y. K.; Oh, Y.-K., Cell disruption and lipid extraction

ACS Paragon Plus Environment

Page 10 of 20

Page 11 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

for microalgal biorefineries: A review. Bioresour. Technol. 2017.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figures captions Figure 1. Schematic of astaxanthin extraction from H. pluvialis using SHS. Figure 2. Efficiencies of astaxanthin extraction from wet H. pluvialis using DMCHA at microalgae/DMCHA ratios of 5 mg mL-1, 100 mg mL-1, and 250 mg mL-1 for 1 h, 3 h, 6 h, and 24 h. Figure 3. SEM images of H. pluvialis (a) before and (b) after 24 h of extraction with DMCHA.

ACS Paragon Plus Environment

Page 12 of 20

Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Figure 1. Schematic of astaxanthin extraction from H. pluvialis using SHS.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Efficiencies of astaxanthin extraction from wet H. pluvialis using DMCHA at microalgae/DMCHA ratios of 5 mg mL-1, 100 mg mL-1, and 250 mg mL-1 for 1 h, 3 h, 6 h, and 24 h.

ACS Paragon Plus Environment

Page 14 of 20

Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Figure 3. SEM images of H. pluvialis (a) before and (b) after 24 h of extraction with DMCHA.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only

Dimethylaminocyclohexane was used to extract astaxanthin from wet H. pluvialis with 80% water content, and the extraction efficiency reached 87.2%.

ACS Paragon Plus Environment

Page 16 of 20

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Figure 1. Schematic of astaxanthin extraction from H. pluvialis using SHS. 228x140mm (144 x 144 DPI)

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2: Efficiencies of astaxanthin extraction from wet H. pluvialis using DMCHA at microalgae/DMCHA ratios of 5 mg mL-1, 100 mg mL-1, and 250 mg mL-1 for 1 h, 3 h, 6 h, and 24 h. 297x207mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 18 of 20

Page 19 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Figure 3: SEM images of H. pluvialis (a) before and (b) after 24 h of extraction with DMCHA. 336x235mm (72 x 72 DPI)

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only Dimethylaminocyclohexane was used to extract astaxanthin from wet H. pluvialis with 80% water content, and the extraction efficiency reached 87.2%. 140x92mm (144 x 144 DPI)

ACS Paragon Plus Environment

Page 20 of 20