One-pot, simultaneous cell wall disruption and complete extraction of

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One-pot, simultaneous cell wall disruption and complete extraction of astaxanthin from Haematococcus pluvialis at room temperature Muhammad Irshad, Aye Aye Myint, Min Eui Hong, Jaehoon Kim, and Sang Jun Sim ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.9b02089 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 21, 2019

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ACS Sustainable Chemistry & Engineering

One-pot, simultaneous cell wall disruption and complete extraction of astaxanthin from Haematococcus pluvialis at room temperature Muhammad Irshada,§, Aye Aye Myintb,§, Min Eui Hongc, Jaehoon Kima,b,d,*, Sang Jun Simc,* aSKKU

Advanced Institute of Nano Technology (SAINT)

2066, Seobu-Ro, Jangan-Gu, Suwon, Gyeong Gi-Do 16419, Republic of Korea bSchool

of Mechanical Engineering, Sungkyunkwan University

2066 Seobu-Ro, Jangan-Gu, Suwon, Gyeong Gi-Do 16419, Republic of Korea cDepartment

of Chemical and Biological Engineering, Korea University

Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea dSchool

of Chemical Engineering, Sungkyunkwan University

2066 Seobu-Ro, Jangan-Gu, Suwon, Gyeong Gi-Do 16419, Republic of Korea

§Equally

contributed

*Corresponding

authors: [email protected] (Jaehoon Kim), Sang Jun Sim

([email protected])

ACS Paragon Plus Environment

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Abstract The extraction of diverse thermolabile, but highly value-added chemicals entrapped in the robust cell wall of algal biomass has remained a remarkable challenge. This study investigated the onepot, simultaneous cell wall disruption and extraction of bioactive astaxanthin from the cysts of Haematococcus pluvialis. During the extraction, wet grinding in the presence of ethanol caused the intimate contact of the solvent with the ruptured cells, resulting in almost complete astaxanthin recovery of 31.6 mg per g of dried H. pluvialis (>99% recovery) and significantly high extraction yield of 46.9 wt% at room temperature (around22 oC) and atmospheric pressure in a very short time (≤30 min) under mild milling condition (≤200 rpm). The one-pot method allowed the use of other types of generally recognized as safe solvents such as acetone, ethyl acetate, hexane, and isopropyl alcohol. The antioxidant test indicated that the crude extracts were highly active in scavenging radicals. Thus, our one-pot method was an eco-friendly and economical extraction approach for the complete recovery of astaxanthin in a simple, rapid, lowtemperature, and low-pressure manner that minimized the energy required for astaxanthin extraction and suppressed its degradation. Keywords: Haematococcus pluvialis, one-pot method, cell wall disruption, extraction, astaxanthin, antioxidant activity, GRAS solvent


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INTRODUCTION The utilization of microalgae in food, chemical, and fuel applications has become a highly promising and viable option for biorefineries to offset the overall biofuel and biochemical production costs by producing high value-added products.1-2 Microalgae have a remarkable photosynthetic potential to reduce the negative environmental impacts associated with global warming.3-5 Freshwater microalgae such as Haematococcus pluvialis are one of the most promising feedstocks for being a potent rich natural source of astaxanthin (C40H52O4, 3,3′dihydroxy-β,β-carotene-4,4′-dione). Astaxanthin is a red secondary ketocarotenoid containing thirteen conjugated double bonds and has strong antioxidant activity.6-7 Therefore, natural astaxanthin extracted from H. pluvialis can be used for various therapeutic applications, including cardiovascular diseases, immune system dysfunction, cancer, inflammatory infections, aging, and muscle soreness.8-11 Astaxanthin is expected to achieve 25% market shares of the total carotenoid revenue (USD 1.52 billion) by 2021.12 At present, because of the highly expensive photobioreactors for cultivating H. pluvialis, the natural astaxanthin (market value, 2500–7000 $/kg) accounts for less than 1% of the commercially available product, whereas its synthetic counterpart from the petrochemical origin (market value, 1000 $/kg) occupies 95% of the market share.13-14 Because of the inherent difference in the ratios of stereoisomers in synthetic and natural astaxanthin (ratio of 1:2:1 and 1:1:22 for 3R,3′R; 3R,3′S; and 3S,3′S stereoisomers in synthetic and natural astaxanthin, respectively,15-16 see Figure S1a) and the extraordinary bioactivity of the 3S,3′S isomer,17 natural astaxanthin exhibits considerably higher bioavailability than the synthetic counterpart after dietary supplementation.18 Thus, synthetic astaxanthin is mainly used as a feed additive in aquaculture only.


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The highest accumulation of 3S,3′S astaxanthin is noted in the microalgal cysts of H. pluvialis, which makes it the most appealing feedstock for the extraction of natural astaxanthin and biorefinery for producing fuels from the extracted residue.19 H. pluvialis can concentrate up to 1–5 wt% astaxanthin of its dry weight at the aplanospore stage under unfavorable condition (e.g., elevated temperature, high salinity, nitrogen deficiency, and absence of light).9, 20 However, stimulating conditions, which aid in the accumulation of astaxanthin, also induce the formation of a highly robust, acetolysis-resistant circumferential wall around the cyst cells, which consists of sporopollenin material (algaenan), proteins, cellulose, and carbohydrates.21-22 The thickness of the robust cell wall is in the range of 1.8–2.3 μm,21, 23 which renders the extraction of astaxanthin difficult. Astaxanthin accumulates in the form of lipid droplets near the nucleus inside the cytoplasm of cyst cells. In the cells, only 5% of astaxanthin exists in the free form, whereas the remaining is present in the form of fatty acid monoesters and diesters that are bonded to astaxanthin.24 Effective recovery of free astaxanthin and its ester derivatives from the cysts of H. pluvialis requires the efficient rupturing of the highly robust cell wall. Therefore, the disruption of the cell wall and extraction of astaxanthin from the mature cysts of H. pluvialis without losing the bioactivity of astaxanthin remains a remarkable challenge for the ultimate realization of microalgal biorefineries. Over the last few decades, numerous conventional and emerging microalgal technologies for cell wall disruption and astaxanthin extraction have been developed for the efficient recovery of astaxanthin from the mature cysts of H. pluvialis;25-38 some previous studies are summarized in Tables S1–S2. The largest possible amount of astaxanthin was recovered from H. pluvialis by using approaches involving two steps: mechanical, chemical, thermal or biological disruption of the cell well and subsequent solvent extraction.25, 27, 29-30, 32-33, 35-46 If cyst cells are completely


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disrupted during the first step, medium-to-high recovery of astaxanthin in the range of 8–23 mg/g from H. pluvialis was achieved in the second extraction step because of the high solubility of astaxanthin in some organic solvents (e.g., dichloromethane, acetone, and hexane) or green solvents (e.g., supercritical carbon dioxide with the co-solvent dimethyl ether).25, 27, 29, 32-33, 37-38, 41, 46-47

Therefore, complete cell wall disruption, which is the key step for achieving high

astaxanthin recovery, was achieved using various types of pretreatment techniques such as mechanical approaches (e.g., bead beating, high-speed and high-pressure homogenization, grinding, and ultrasound-assisted extraction),25,

27, 29, 32-33, 36-38, 41, 46-47

high-pressure and high-

temperature approaches (e.g., hydrothermal treatments),30, 35, 46 chemical approaches for the lysis of H. pluvialis cells (e.g., acid, base, and solvent),33, 37-38, 42, 46, 48 enzymatic lysis,45, 49 dormancyrelease cell septa rupture33, ionic liquids39, 43, and switchable hydrophilicity solvents.40 Although two-step approaches could guarantee a high-yield astaxanthin recovery, high-energy consumption during the pretreatment step associated with mechanical grinding, high cost of some chemicals, long pretreatment and extraction time, the risk of astaxanthin bioactivity loss, and incomplete cell wall rupturing need to be carefully addressed before their implementation at a practical scale. Unlike the mechanical approaches, the use of either chemical or enzymatic lysis could be an energy-efficient alternative extraction method. However, most of the chemical and enzymatic approaches resulted in low astaxanthin recovery values in the range of 1–29 mg/g (see Table S1).37-40,

46,

49

Other drawbacks associated with the chemical methods include

contamination, mutation effect, and risk of astaxanthin degradation on contact with chemicals.5051

To date, only a few one-pot extraction strategies for simultaneous cell wall disruption and

extraction have been reported (see Table S2); as expected, the astaxanthin recovery yields obtained using these methods were low (1–27 mg/g).26, 40, 42, 49, 52-53


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Most previous studies focused on high-yield recovery of astaxanthin, but relatively less attention has been paid to reduce the time required for the pretreatment and extraction of astaxanthin. Astaxanthin is known to be highly sensitive toward physical or chemical degradation once it is exposed to air, temperature, ultrasound, microwave, and light radiations5456;

for example, even very low temperatures (50–60 °C) were found to adversely affect the

function of astaxanthin.55 Mitigating the risk of degradation during the recovery of astaxanthin requires the development of a rapid, simple, and efficient pretreatment and extraction technique under mild conditions (e.g., inert environment, low-pressure, low-temperature, short-term pretreatment, and extraction). In this study, we showed that one-pot, simultaneous cell wall disruption and complete extraction of astaxanthin along with its fatty acid ester derivatives from the cysts of H. pluvialis can be achieved in a very short time by using simple wet ball milling in the presence of any appropriate extraction solvent. During the wet ball milling, the robust cell wall of H. pluvialis was effectively disrupted, and astaxanthin and its ester derivatives were simultaneously extracted completely in the solvent. Thus, the total astaxanthin recovery was in the range of 31.6 mg/g of dried H. pluvialis; this value was remarkably higher than those obtained using most of the previously reported two-step,30, 32, 34, 36-41, 43-45, 47 as well as one-step approaches (see Tables S1– S2).26, 40, 42, 49, 52-53 In addition, the red-orange colored pigments in H. pluvialis were completely extracted. This study aimed to develop an eco-friendly and economical extraction approach for the complete recovery of astaxanthin by using a one-pot, simple, rapid, low-temperature, and low-pressure method that can minimize the energy required for astaxanthin extraction and suppress its degradation. To the best of our knowledge, no one-pot, simultaneous cell wall disruption and extraction of astaxanthin approach has such high efficiency. The effects of ball


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milling time and energy as well as generally recognized as safe solvents (ethanol, acetone, nhexane, ethyl acetate, and isopropyl alcohol) on the extraction and antiradical activity of the extracts were investigated. Radical scavenging activities of the extracts obtained using different solvents were assessed using two commercial antioxidant standards and two assays methods.

EXPERIMENTAL Algal strain, solvents, reagents, and chemical standards The H. pluvialis strain (NIES-144, wild-type) was purchased from the National Institute for Environmental Studies (Tsukuba, Japan). H. pluvialis cells were grown fully until a high amount of astaxanthin was accumulated at its aplanospore stage in an outdoor microalgal culture system, which was installed in a combined heat and power plant, located in South Korea. An industrial flue gas emitted from the combustion of liquefied natural gas was coupled to the culture system for heating. A detailed description of the flue gas composition, culture medium composition, and photobioreactor design is available in our previous studies.20, 57 The harvested H. pluvialis cyst cells were lyophilized at –55°C by using a freeze dryer (model FDB-5503; Operon, South Korea). High-purity argon (Ar) gas (≥99.999%) was purchased from JC Company (South Korea). The solvents used in this study, including dichloromethane (DCM; purity, ≥99.8%), acetone (purity, ≥99.9%), methanol (purity, ≥99.8%), hexane (purity, ≥95.0%), and ethyl acetate (EA; purity, ≥99.8%), were purchased from Daejung Chemicals & Metals (South Korea). Ethyl alcohol (purity, ≥99.99%) and isopropyl alcohol (IPA; purity, ≥99.9%) were purchased from Burdick & Jackson (USA). De-ionized (DI) water was purchased from Samchun Pure Chemical (South Korea). Potassium persulfate (purity, ≥95%) and sodium hydroxide (purity, ≥97%); astaxanthin standard (purity, ≥97% from H. pluvialis); 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid


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diammonium salt (ABTS; purity, >98%); 1,1-diphenyl-2-picrylhydrazyl (DPPH; purity, ≥95%); gallic acid (purity, >97%); FolinCiocalteu phenol reagent (concentration: 2 N); and Trolox (6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; purity, >97%) were purchased from Sigma Aldrich (USA). One-pot, simultaneous cell wall disruption and extraction The degradation of astaxanthin in the dried H. pluvialis was prevented by storing the dried sample at –80°C until further use. One gram of dried H. pluvialis sample, 70 g of zirconia balls, and 10 g of experimentally desired solvent were introduced in the vessel of a planetary-type micro ball mill apparatus (PULVERISETTE 7; premium line, FRITSCH, Germany). Potential contamination during the one-pot grinding and extraction was avoided by using zirconia balls with a diameter of 5 mm as the grinding media for their good ductility and wear-resistant nature. After the dried H. pluvialis, zirconia balls, and solvent were loaded, the ball mill chamber was purged with high-purity Ar gas to maintain an inert gas environment during the cell disruption and astaxanthin extraction process. For each extraction solvent, cell disruption was performed at varying time intervals (5–30 min) and rotational speeds (100–200 rpm). The ball mill apparatus was coupled with a forced-air cooling jacket to maintain the internal temperature below 25 °C during the rotation. Furthermore, short time run (5 min) and equal cooling pauses was used to remove heat from the ball milling chamber. After an experimentally desired ball milling period, the chamber was opened, and the mixture was collected in a beaker. The extracts were completely recovered by washing the grinding media and inner dome with 100 mL of the same solvent used for the extraction. The mixture was then filtered on a pre-weighed filter paper, and the solid residue remaining on the filter paper was dried in a conventional oven at 80 °C for 12 h.


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The wet grinding process was compared with conventional dry grinding by introducing the same ratio of grinding media-to-H. pluvialis into the ball mill chamber without adding any solvent. The dry ground sample was Soxhlet extracted using acetone at 65 °C for 12 h. Liquid extract yield and astaxanthin quantification After the one-pot cell wall disruption and extraction, the changes in the mass of the solid sample were calculated using a high-accuracy digital mass balance (model X124 Explorer series; OHAUS, USA). The overall extraction yield of the extractives present in the dried H. pluvialis was calculated using equation 1. Extract yield (wt%) =

Weight of dried 𝐻. 𝑝𝑙𝑢𝑣𝑖𝑎𝑙𝑖𝑠 ― Weight of solid residue Weight of dried 𝐻. 𝑝𝑙𝑢𝑣𝑖𝑎𝑙𝑖𝑠

× 100%

(1)

The amount of astaxanthin in the extract was calculated using high-performance liquid chromatography (HPLC). Astaxanthin (mg) =

HPLC astaxanthin concentration (ppm) × Extract mass (g) × 1000 mg g

(2)

In equation 2, astaxanthin (mg) is the total mass of astaxanthin in the extract, HPLC astaxanthin concentration (ppm) is the astaxanthin concentration measured in the extract solution by using HPLC, and extract mass is the total mass of the extract solution (g). The astaxanthin recovery yield was quantified using equation 3. Astaxanthin recovery (mg/g) =

Weight of extracted astaxanthin (mg) Weight of dried 𝐻. 𝑝𝑙𝑢𝑣𝑖𝑎𝑙𝑖𝑠 (g)

(3)

Thermogravimetric analysis The moisture and inorganic contents of the dried H. pluvialis were measured by conducting thermogravimetric analysis (TGA) by using a Q50TGA equipment (TA Instruments, USA) at a temperature range of 30 to 800 °C. The heating rate was set at 8 °C/min, and the gas flow rate


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was fixed to 60 mL/min. The results of proximate analysis performed on freeze-dried H. pluvialis by using the TGA profiles (as shown in Figure S2) are listed in Table S3. Chromatographic analysis The concentration of astaxanthin in the different organic solvents after each extraction was quantified in duplicate by using a reverse-phase HPLC method.25 Alliance HPLC (model e2695; Waters, USA) equipped with a modal 87E UV-Vis detector was used for the quantification of astaxanthin. The YMC-C30 reversed-phase carotenoid column (250 mm length × 4.6 mm inner diameter × 5 µm particle size; YMC Co., Japan) was used. The temperature of the column was maintained at 30 °C during the analysis. A diluted sample was passed through a 0.45 μm PTFE syringe filter before injecting into the sample vial. The composition of the mobile phase was adjusted to solvent A (acetone:water in an 84:16 ratio, v/v) and solvent B (acetone:water in a 97:3 ratio, v/v). A good separation of free astaxanthin from its ester derivatives and other carotenoids was achieved by programming the mobile phase elution as follows: isocratic elution with solvent A for the first 10 min, followed by gradient elution from 0% to 100% with solvent B for the next 110 min, and then isocratic elution with solvent A again up to 120 min. The flow rate of the mobile phase was set at 1 mL/min, and the sample injection volume was set at 10 µL by using an autosampler. The UV-Vis detector recorded the absorbance at 480 nm. A standard calibration curve with a regression factor of R2 = 0.9997 was plotted using the astaxanthin standard in DCM by using varying concentrations in the range of 80–656 µg/L. The free astaxanthin content in the extract was calculated using the retention time for the astaxanthin standard, and the other peaks in the HPLC chromatograms, attributed to the ester derivatives of astaxanthin and other types of carotenoids, were assigned using relevant literature.58


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Changes in coloration and morphology The morphological changes in H. pluvialis cyst cells before and after the extraction were observed using a field-emission scanning electron microscope (FE-SEM; JSM7500F; JEOL, Japan). The residual pigments in the solid residue after the extraction were estimated, and the extraction efficiencies were compared by quantifying the changes in coloration of the solid residue by processing digital camera images by using MATLAB software (2012b) to calculate the RGB pixel values. The residual pigments were estimated by measuring the normalized red contents by using the RGB values according to the method shown in Supporting information.35 The antiradical activity of the extracts Because of the existence of double bonds and terminal phenolic groups in astaxanthin, it acts as an antiradical by either electron or hydrogen atom transfer for radical scavenging.59-61 In this study, two control antioxidants (gallic acid and Trolox) and two assay methods (DPPH• and ABTS•+) were used to test the antioxidant activity of the crude extracts without further saponification and purification. The FolinCiocalteu reagent was used to determine the phenolic contents in the extracts. The antioxidant activity of the astaxanthin extracts was compared with that of the standard free astaxanthin purchased from Sigma-Aldrich. DPPH• scavenging activity The antioxidant activity of the extracts was determined by analyzing their scavenging activity toward DPPH• and comparing with two types of standard antioxidant solutions (Trolox and standard free astaxanthin) at a concentration of 1 mM, according to a previously described method with slight modification.62 Briefly, a homogeneous 0.15 mM DPPH• stock solution was prepared in methanol and kept aside for 20 min. A volume of 100 μL of each crude extract


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obtained using different organic solvents was separately mixed with the 100 μL DPPH• solution. Next, the mixture was incubated at 25 °C for 30 min in the dark. The absorbance of the mixture was read at 517 nm by using a microplate spectrophotometer (Power wave X 340; BioTeK Company, South Korea). The radical scavenging activity was determined as the percentage of DPPH• discoloration by using equation 4. Radical scavenging activity (%) =

(

ADPPH ― (AS ― ASC) ADPPH

) × 100%

(4)

where ADPPH, AS, and ASC represent the absorbance of the DPPH–methanol solution, the mixture of DPPH–methanol solution and the extract, and the pure extract, respectively. The IC50 value, a measure of the inhibition concentration to scavenge DPPH• by 50%, was calculated using linear interpolation. A graph of the radical scavenging activity of each extract was plotted by varying the concentrations of the extracts in the range of 40–700 µg/mL. In addition, a standard curve of Trolox was drawn by varying its concentration in the range of 4–100 µM, and the DPPH• values of the extracts were expressed as μmol of Trolox equivalent (TE) per g of extract. The higher the DPPH• values, the higher is the antioxidant activity and vice versa. ABTS•+ scavenging activity The ABTS•+ scavenging activity of the extracts was measured according to the method described previously63 with some modifications. Briefly, ABTS•+ stock solution, which consisted of 7 mM ABTS•+ and 2.45 mM potassium persulfate in DI water, was kept for 16 h at 25°C to allow the completion of radical generation in the absence of light. The ABTS•+ stock solution was then diluted with DI water to achieve the absorbance of approximately 0.70 at 734 nm and used as the working solution. A volume of 30 μL of the extract was mixed with 1.47 mL of the ABTS•+ working solution and incubated for 6 min at 25 °C in the dark. The absorbance of the


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mixture was read at 734 nm against a blank of DI water by using a microplate spectrophotometer. The radical scavenging activity was determined as the percentage of ABTS•+ discoloration by using equation 5. Radical scavenging activity (%) =

(

AABTS ― (AS ― ASC) AABTS

) × 100%

(5)

where AABTS, AS, and ASC represent the absorbance of the ABTS–methanol solution, the mixture of DPPH–methanol solution and the extract, and the pure extract, respectively. The ABTS•+ values for the extracts were measured using a standard curve plotted for Trolox concentration in the range of 8–250 µM, and the data were expressed as μmol of TE per g of the extract. The larger the ABTS•+ values, the higher is the antioxidant activity and vice versa. Total phenolic contents The total phenolic contents of the extracts were measured using different types of solvents and a well-known assay procedure reported in the literature.64 Briefly, a solution of sodium carbonate in DI water (10 wt%) was prepared. A volume of 0.25 mL of each crude extract, sodium carbonate solution, and FolinCiocalteu reagent were mixed together. Next, 2.5 mL of DI water was added to the mixture solution, and the samples were incubated at 25 °C for 1 h. The absorbance of the samples was recorded at 725 nm by using a UV-3600 UV-Vis spectrophotometer (Shimadzu, Japan). A calibration curve with R2 = 0.995 was constructed by varying the gallic acid concentrations in the range of 3–56 µg/mL. The phenolic content of each extract was expressed as mg of gallic acid equivalents (GAEs) per g of the extract obtained from dried H. pluvialis.


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Statistical analysis for significance The reproducibility of the experiments performed in this study was confirmed by performing the extractions of each ball milling duration in each solvent in triplicate and averaging the values within ±5% standard deviation. The antiradical activity of each extract tests was performed in triplicate and average values with standard deviations were reported.

RESULTS AND DISCUSSION Comparison of the one-pot method with the two-step method A schematic of the one-pot, simultaneous cell wall disruption and astaxanthin extraction from the red cyst of H. pluvialis is shown in Figure 1. In this one-pot approach, dried H. pluvialis and extraction solvent were introduced together into the ball mill chamber. Therefore, the pretreatment for rupturing cell wall, which has been adopted in most previous studies as an elementary independent step before extraction,25,

27, 29-30, 32-33, 35-43, 45-47

can be avoided. In

addition, in the one-pot approach, astaxanthin recovery and total liquid extraction yield were enhanced significantly in a short time under mild milling conditions; at 200 rpm, the astaxanthin recovery was 27 mg/g (>82% recovery) and 31.6 mg/g (>99% recovery), which were substantially higher than those reported previously, at considerably shorter processing times of 10 and 30 min, respectively, (see Tables S1–S2 Supporting information). The one-pot method developed in this study and two-step extraction strategies that have been used in most of the previous studies mainly differ in the approach used to rupture the cells and to extract the solutes. In the two-step method, the dried biomass was ground initially, and a subsequent extraction was performed using Soxhlet, supercritical fluid, or other extraction methods; in contrast, in the onepot method, wet grinding and extraction were performed simultaneously. The intimate contact of


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the solvent with ruptured cells during the one-pot grinding and extraction allowed high-yield astaxanthin extraction in a short processing time and at room temperature, unlike that obtained using dry grinding and subsequent extraction. Therefore, the one-step approach could be considered as a highly energy-efficient, rapid, simple, and safe method for the high recovery of astaxanthin from dried H. pluvialis.

Figure 1. Schematic representation of the one-pot, simultaneous cell wall disruption and extraction of astaxanthin from H. pluvialis.

During the dry ball milling of the red cysts of H. pluvialis, a cell debris cake is typically formed, resulting in the formation of a coating of the cell debris layer on the wall of the chamber and on the zirconia balls; for example, after the 10 min of grinding of dried H. pluvialis at 200 rpm, extensive cake formation and coating layers were observed (Figure 2a). In principle, mechanical disruption during ball milling typically causes a strong shear force on the cell wall and ultimately peels off the cell wall of the cyst cells. However, the caking and coating


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phenomena, which are caused by preferential agglomeration of the H. pluvialis debris, can result in a leveling effect on the cell wall disruption and astaxanthin recovery. In other words, once the cake layer is generated, further grinding becomes less effective to rupture the entrapped and hidden cyst cells under the cake bed. Thus, the leveling effect can cause a significant increase in the pretreatment duration and a decline in the grinding efficiency. Therefore, highly energyintensive continuous ball milling for an extended time is required to achieve complete cell wall disruption, which remarkably increases the cost for the overall process. In addition, the caking and coating might lead to frequent interruptions of the pretreatment process to remove the sticky cell debris layers from the grinding media and the dome of the ball mill. In the dry grinding of H. pluvialis, thick and sticky slurry is produced by the residual water in the cyst cells, eluted lipids, and disrupted cell debris (Figure 2a). The slurry leads to the formation of a cake layer on the walls of the ball mill chamber and the surface of balls. Further, “compaction” of the cake layer is caused by the continuous impact of the balls, which might result in the loss of the bioactivity of the eluted astaxanthin: the energy gained by the caking mixture from the continuous hitting of the balls at a high rotational speed can lead to the degradation of astaxanthin through the excitation of the unsaturated double bonds. Therefore, extended grinding time, unavoidable frequent interruptions of the pretreatment process, requirement of an additional technique for recovering of the cell debris layer, and degradation of astaxanthin associated with dry grinding make the process highly energy-intensive, inefficient, costly, and unattractive to be implemented at a practical scale. In addition, in the subsequent extraction step, the use of a large volume of solvents (e.g., organic solvents, supercritical carbon dioxide, and dimethyl ether) and prolonged extraction times (1–24 h; Table S1) are typically required for recovering the eluted free astaxanthin and its ester derivatives.


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Figure 2. (a) Caking during the dry grinding of dried H. pluvialis cyst cells for cell wall disruption and (b) No caking observed during the one-pot, simultaneous cell wall disruption and extraction.

The astaxanthin recovery in the one-pot, simultaneous cell wall disruption and astaxanthin extraction approach developed in this study has numerous advantageous over the two-step method: first, during the wet ball milling, the cyst cell walls, which are disrupted by the shear force, can be directly solubilized in an appropriate solvent, which can suppress the formation of cake and coating layers on the surface of the chamber and balls (Figure 2b). Second, unlike the subsequent extraction in the two-step method (e.g., Soxhlet), the one-pot approach utilizes a very small amount of solvent, reducing the energy required to recover the dried astaxanthin powder


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by solvent drying. For example, 250 mL acetone was used in the Soxhlet extraction of the disrupted cell wall debris at a siphon rate of 4/h, whereas only 100 mL of acetone was found to be sufficient in the one-pot method for the extraction and complete washing by using the same amount of dried H. pluvialis. Third, the time required for cell wall disruption and extraction in the one-pot method was significantly reduced compared to that required in the two-step method, which could reduce the total operating cost. The total processing time required for the pretreatment and extraction in the two-step method is typically more than 12 h, whereas 30 min was found to be sufficient for the complete extraction of astaxanthin in the one-pot method. Fourth, in the one-pot method, effective cell wall disruption and extraction can be performed at a very low rotational speed (100–200 rpm) and low temperature (below 25 °C), which can suppress the risk of degradation of the highly thermolabile astaxanthin. The electron-rich conjugated double-bond structure of astaxanthin renders it highly sensitive to physical and chemical degradation once it is exposed to air, high temperature, and light radiation

54-55, 65;

for

example, high temperature inhibits astaxanthin function, as it can be degraded with first-order reaction kinetics even at very low temperatures (50–60°C).55 Lastly, the one-pot method could be performed using many low-toxicity solvents such as ethanol, acetone, n-hexane, IPA, and EA, because the requirement of low-boiling-point solvents to suppress the degradation of astaxanthin during a typical solvent-based extraction (e.g., Soxhlet) is overcome. Therefore, the one-pot method for simultaneous cell wall disruption and extraction is more energy-efficient, more flexible, more cost-effective, simpler, and safer for the recovery of astaxanthin compared to the two-step methods. A comparison of astaxanthin recovery and total extraction yield of the two-step and one-pot methods is shown in Figure 3. In the pretreatment process of the two-step method, the disruption


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of dried H. pluvialis cell wall was performed at 200 rpm for 10 min; subsequently, the pretreated sample was Soxhlet-extracted in acetone for 12 h. The two-step method resulted in astaxanthin recovery yield of 21.0 mg/g of dried H. pluvialis and liquid extract yield was 32.0 wt%. In the one-pot method, the dried H. pluvialis, zirconia balls, and acetone with H. pluvialis-to-solvent ratio of 1:10 w/w were introduced in the vessel of the extracting mill together and processed at 200 rpm for 10 min. The one-pot method resulted in a significantly higher astaxanthin recovery yield of >27 mg/g of dried H. pluvialis and extraction yield of 42.0 wt% compared to that of the two-step method under identical ball milling time and rotational speed. In case of using a high rotational speed of 300 rpm in one-pot method, a high extraction yield of 45.9 wt% could be achieved in a very short time of 6 min (data are not shown). The time course efficiency of cell wall disruption and extraction was considerably higher in the one-pot method than in the twostep method. The red color of the cell debris collected after the extraction is shown in Figure 3b. The cell debris collected after the two-step method still contained some pink coloration, suggesting that all the red-orange colored pigments were not completely extracted. In sharp contrast, the color of the cell debris collected after the one-pot method was only light greenish, indicating efficient extraction of the red-orange colored pigments, including astaxanthin, except for some remaining chlorophylls, which had low solubility in acetone.66-67 Furthermore, the SEM images of the solid residues confirmed the high degree of cyst cell breakage in the one-pot method compared to that in the two-step method, which justified a correlation between the high extraction yields and the high degree of cell wall disruption. The high extraction and astaxanthin recovery values observed in the one-step method are attributed to the effective contact of the solvent with extractables and agitation generated by the ball milling. The efficient the cell wall


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disruption during the one-pot method could occur because of the soaking of solvent in the solid matrix can lead to change textural properties of the cell wall.

Figure 3. (a) Extraction yield and astaxanthin recovery at 200 rpm by using one-pot (10 and 20 min) and two-step methods (10, 30, and 60 min). The control in Figure 3a indicates extraction yield and astaxanthin recovery of the freeze-dried H. pluvialis using Soxhlet extraction in acetone for 12 h without the ball milling treatment. (b) Images of the cell debris obtained using a digital camera and scanning electron microscope: (i) dried H. pluvialis cyst cells before pretreatment, (ii) two-step extraction (10 min), and (iii) one-pot, simultaneous cell wall disruption and extraction (10 min).


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The HPLC chromatogram of the Soxhlet-extracted solution from the untreated H. pluvialis showed an extremely low extraction yield and total astaxanthin recovery of 80%) of the total carotenoids, and the minor pigments included β-carotene (approximately 1%) and canthaxanthin (approximately 5.1%)

10.

Therefore, the red appearance

of H. pluvialis is was mostly attributed to the presence of astaxanthin in its free form and its esterified derivatives. The astaxanthin derivatives are C16–C18 fatty acid monoesters and diesters, the nature of which is either very weakly acidic or neutral (Figure S3d). Thus, the presence of these fatty acid mono- and diesters might not alter the red color intensity of astaxanthin.69 Therefore, the change in the solid residue from dark red to colorless or light gray after the extraction indicates the extent of cell wall disruption and extraction of the red pigments.


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The degree of reduction in the red color of cell debris, calculated using MATLAB, can provide a direct prediction of the extraction efficiency.30, 35 The pigments in the dried H. pluvialis before processing were considered as control, and the color of the solid residue collected after the extraction was expressed as a normalized red content by using equation S1, and the results are shown in Table S4. Converting the 8-bit pixel values of the digital images of the control and almost colorless solid residue into RGB values requires that maximum and zero pigments, respectively, are shown as normalized red content to serve as an indicator for the time-course extraction efficiency (Table S5). Residual red content in the solid residue was correlated well with the experimentally determined astaxanthin recovery (Figure 4). At the very beginning of the one-pot cell wall disruption and extraction for 5 min, a remarkable reduction in the red content of the solid residue was observed, indicating that the dried cyst cells were ruptured effectively. A further increase in processing time gradually decreased the red contents after 30 min of extraction. The rotational speed of 200 rpm was found to be more effective to reduce the normalized red contents up to >80% in only 10 min compared to those remaining at 100 rpm. The reduction in red color intensity indicated that the total astaxanthin recovery of 21 mg/g of the dried H. pluvialis was achieved in a very short time of 5 min ball milling at 200 rpm, which corresponds to approximately 65% astaxanthin present in the cysts of H. pluvialis. When the time was extended to 10 min at 200 rpm, approximately 82% of astaxanthin was recovered. When the time of wet ball milling was further increased to 30 min, an extremely high astaxanthin recovery of >31.6 mg/g (approximately 99%) was achieved. The astaxanthin recovery at a very low rotational speed of 100 rpm was found to be consistently lower than that at 200 rpm at all the times investigated. The SEM images of the debris collected after the extraction confirmed more effective cell wall disruption at 200 rpm compared to that at 100 rpm for the same duration


24

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(Figure S4). This indicates that the highly robust cell wall required either longer time or high shear force for the complete disruption of the cysts.

Figure 4. (a) Effect of ball mill speed and time on cell wall disruption, residual red contents of solid residue, total astaxanthin recovery in the liquid extract. Digital camera images of the cyst cell prior to extraction (before in Figure a) and cell debris after the one-pot, simultaneous cell wall disruption and extraction for 5–30 min at (b) 200 rpm and (c)100 rpm.


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Effect of solvent on the extraction efficiency and astaxanthin recovery The choice of solvent, one of the most important factors in the field of separation and extraction , needs to be determined based on the solubility of the target solute, toxicity of the solvent, and undesirable changes in bioactivity of the solute.70-71 The solvent should be less or non-toxic and have minimal effect on the stability of highly vulnerable bioactive species of interest, such as astaxanthin. The solubility of astaxanthin depends on its isomeric structure and solvent polarity;72-73 for example, the solubility of all-E-astaxanthin was the highest in acetone (0.16 g/L), followed by that in ethanol (0.008 g/L) and hexane (0.003 g/L), whereas an extract having 63% Z-isomers exhibited considerably higher solubility in acetone (5.7 g/L) and ethanol (5.6 g/L) than in hexane (1.54 g/L) compared to an extract having all-E-astaxanthin.72 At the mature cyst stage, astaxanthin in H. pluvialis mainly exists in an all-E-astaxanthin form, which has a very high tendency to isomerize to Z-isomeric form in organic solvents for steric purposes (Figure S1b).58, 74 Unlike Soxhlet extraction, one-pot cell wall disruption and extraction was highly versatile in the use of various types of solvents for the extraction of thermolabile astaxanthin and other pigments and lipids. In this study, five different solvents—ethanol, acetone, n-hexane, IPA, and EA, which have been frequently used in food and pharmaceutical industries as extraction and separation solvents for bioactive natural products,70-71, 75-76 were tested in the one-pot cell wall disruption and extraction method. The effect of solvent on the extraction yield and astaxanthin recovery at 25 °C, 1 atm pressure, and 200 rpm is shown in Table 2 and Figure 5, respectively. The extraction yield was in the highest in ethanol (46.9 wt%), followed by that in acetone (44.8 wt%) and EA (44.7 wt%), IPA (43.5 wt%), and hexane (39.5 wt%); further, astaxanthin recovery was of the highest in ethanol (31 mg/g), acetone (31.6 mg/g), and EA (31.2 mg/g), followed by


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that in IPA (24 mg/g) and hexane (17 mg/g). The higher extract yield in ethanol, acetone, and EA than in IPA and hexane could be because of the ability of the former solvents to solubilize both polar and non-polar parts of the extracts presumably due to their polarity effect. At the aplanospore stage of H. pluvialis, the fatty acids comprise neutral lipids (~52%), phospholipids (~21%), and glycolipids (~27%).10 Carotenoids and lipids are lipophilic in nature because of the long chain of hydrocarbons and low content of oxygenated species, and thus they are highly soluble in nonpolar or slightly polar solvents.44 Conversely, some slightly charged lipids (phospholipids) and polar lipids (glycolipids) are more soluble in slightly polar solvents compared to that in non-polar solvents

77.

The high astaxanthin recovery values in ethanol,

acetone, and EA suggested good solutesolvent interactions with these solvents.72 Table 2. Comparison of the liquid extract yield of H. pluvialis obtained after the one-pot, simultaneous cell wall disruption and extraction by using different types of solventsa

Entry

Solvent

Extract yield (wt%)b

Astaxanthin recovery (mg/g)

Solvent polarity index

1

Ethanol

46.9 ± 2.7

30.1 ± 1.5

4.8

2

Acetone

44.8 ± 2.3

30.6 ± 1.5

5.1

3

IPA

43.5 ± 2.1

24.5 ± 1.2

4.0

4

Hexane

39.5 ± 1.9

16.6 ± 0.8

0.0

5

EA

44.7 ± 2.3

29.9 ± 1.5

5.2

a

Conditions: 200 rpm, 1 atm, 30 min, microalgae-to-solvent ratio of 1/10 w/w. of extract per gram of dry sample.

b gram

The experimentally determined astaxanthin recovery in each solvent matched well with the reduction of red contents in the solid residue (Figure 5). The effect of solvents on the


27


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extractability of pigments can be correlated with the changes in the color of the solid residue, indicating different extraction selectivity of the pigments in different solvents with respect to control. For example, the green color of the solid residue collected from acetone, IPA, hexane, and EA can be attributed to the residual colorants existing in H. pluvialis other than astaxanthin (e.g., chlorophyll), which have a low solubility in auxiliary solvents. Ethanol showed the highest solubilizing power for the extraction of green-colored chlorophylls as well as red-colored astaxanthin. Ethanol is known to be a superior solvent to others for the recovery of chlorophylls from different types of algal biomass.66,

78

The possible reason for the high solubility of

chlorophylls in ethanol is the similar polarity of both ethanol and the crude extract from microalgal biomass. Therefore, the one-pot cell wall disruption and extraction method can also be used for the recovery of chlorophylls from green algae by using ethanol as a solvent, without requiring the mandatory pretreatment for the breaking of cell wall.67 One of the most widely used solvents for the extraction of lipids from microalgae for producing biodiesel is hexane79-80 However, surprisingly, the use of hexane in the one-pot cell wall disruption and extraction resulted in slightly lower extraction yield and significantly lower astaxanthin recovery compared to that in the other solvents. This can be attributed to the low solubility of the extract in hexane.81 Except for hexane, all other solvents used in this study can be used to develop a practical-scale carotenoid extraction process owing to their relatively low toxicity.71


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Figure 5. Effect of solvent on the residual red contents of the solid residue and total astaxanthin recovery. Digital camera images of (a) the freeze-dried H. pluvialis cyst cells prior to extraction, and the cell debris collected after the extraction in (b) ethanol, (c) acetone, (d) isopropyl alcohol, (e) hexane, and (f) ethyl acetate in one-pot extraction. The extraction conditions were 200 rpm for 30 min.

Total phenolic contents The total phenolic contents of the extracts, expressed as GAE per g of extract, obtained using different types of solvents are listed in Table 3. The GAE values of the extracts were higher in IPA and ethanol (8.6–10.4 mg GAE/g) than in the other solvents, suggesting good antioxidant


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activity of IPA and ethanol extracts. The GAE value of the standard free astaxanthin was 56 mg of GAE/g of extract. Considering that the crude extract consisted of a small amount of free astaxanthin (5–8 wt% of extract; 1.5–2.7 mg/g of dry sample), mono- and di-esters of astaxanthin, lipids, proteins, and other extractable contents, the GAE values of the crude extract were thought to be high. In fact, the GAE values of the crude extracts obtained in this study were higher than those reported previously for the H. pluvialis extracts and for some other microalgal extracts (1.8–7.7 mg GAE/g).82-84 This can be attributed to the almost complete recovery of astaxanthin and the high extractability of phenolic contents and other pigments by using the onestep method. Conversely, the phenolic content of the extracts was somewhat low in acetone, hexane, and EA in the range of 3–6 mg GAE/g. Depending on the number of polar (hydroxyl groups) and non-polar parts (aromatics rings) in the structure of phenolics, their dissolution varies across different solvents.85-86 Antioxidant activity of the extracts An antioxidant acts as a radical scavenger via two major mechanistic routes depending on its structure and nature of polarity, donating phenolic hydrogen or an electron. The presence of keto (C=O) moieties, hydroxyl (OH) groups on terminal phenols, and thirteen alternate double-bond conjugations is responsible for the high antioxidant activity of astaxanthin that can donate protons to radicals.59-61 The effect of solvent on the antiradical power was investigated by determining the antioxidant activity of the crude extracts, without additional purification and saponification, in different solvents by using the DPPH• and ABTS•+ assays. The antioxidant activity of the extracts was compared with that of Trolox (which is a well-known antioxidant), and the activity was presented in terms of molarity of Trolox equivalents (µmol of TE) per g of


30

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extract (Table 3). In addition, the antioxidant activity of standard free astaxanthin was determined for comparison purpose. The antioxidant activity of the extracts was also expressed as IC50 value, which is the number of antioxidants required to reduce the initial concentration of DPPH• or ABTS•+ to 50%. In principle, the antioxidant activity of an extract in a solution is dependent on the amount and stability of radical scavengers, the nature of solvent used, and the number of radical species.34, 37, 84 The IC50 and Trolox equivalent values of the extracts from H. pluvialis were dependent on the nature of the solvent used (Table 3). Conversely, the antioxidant activity was not necessarily correlated with the astaxanthin recovery values. For example, the DPPH• scavenging activity of the EA extract was the highest, and that of the ethanol extract was the lowest, whereas both the extracts showed similar astaxanthin recovery values (Figure 5). The DPPH• scavenging activity of the extracts was not in the same order as that of the ABTS•+ scavenge activity; the EA extract showed the highest antiradical activity for DPPH•, whereas the acetone extract exhibited the highest ABTS•+ scavenging capability. This indicates that a chemical species that exhibits good antiradical activity for DPPH• does not necessarily show high activity for quenching ABTS•+ and vice versa.87 The difference in the radical scavenging activity depending on the solvent nature and radical species could be attributed to the stereoselectivity of radicals and the dissolution of the extract in diverse solvent systems, which could affect the reaction and quenching rate of the radicals.88 A solvent showing good solubilizing power toward both the extract and radical might bring them close to allow their interaction. In addition, the low stability of the extracted free astaxanthin and its ester derivatives in some organic solvents could result in the low antioxidant activity of radicals.74 The red cysts of H. pluvialis mainly contain an all-E geometrical isomeric form of astaxanthin, which has high sensitivity to transform into 9Z- and 13Z-isomers during extraction in some organic solvents to


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reduce the intramolecular steric hindrance (Figure S1b).58, 74 The 9Z-astaxanthin exhibits higher antioxidant potency against reactive oxygen species than the all-E- and 13Z-geometrical counterparts.89 The antioxidant activities exhibited by the standard free astaxanthin and Trolox were 4 and 28 times higher than those of the crude H. pluvialis extracts, respectively. This is because most of the astaxanthin in the extract was in the long-chain fatty acid ester form. A previous in vitro study showed that free astaxanthin exhibited higher antioxidant activity than its ester derivatives.90 In conclusion, both DPPH• or ABTS•+ radicals were effectively scavenged in all types of solvents used with slightly varying IC50 values, which showed the versatile nature of astaxanthin to exert its antioxidant activity in both hydrophilic and hydrophobic environments when the one-pot method was used.


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Table 3. Total phenolic contents, DPPH•, and ABTS•+ radical scavenging activity of H. pluvialis extracts obtained using different organic solvents after the one-pot, simultaneous cell wall disruption and extraction

Total phenolic DPPH• scavenging activity

ABTS•+ scavenging activity

contents Antioxidants in auxiliary solvent

GAE (mg of GAE/g of extract)a

IC50 (µg/mL)b

Trolox equivalents (µmol of TE/g of extract)c

Trolox equivalents IC50 (µg/mL)

(µmol of TE/g of extract)

Reference Trolox

N.A.

8 ± 0.4

143 ± 7

St. astaxanthind

56 ± 2.8

49 ± 2

1676 ± 84

1562 ± 78

364 ± 18

Ethanol

8.6 ± 0.4

531 ± 27

153 ± 8

3555 ± 177

160 ± 8

Acetone

6.3 ± 0.3

331 ± 17

246 ± 12

3044 ± 152

188 ± 9

IPA

10.4 ± 0.5

304 ± 15

268 ± 13

3371 ± 168

169 ± 8

Hexane

3.2 ± 0.2

242 ± 12

337 ± 17

6274 ± 313

91 ± 4

EA

5.3 ± 0.3

236 ± 12

345 ± 17

4668 ± 233

122 ± 6

H. pluvialis extracte

a Gallic

acid equivalents per gram of extract powder on a dry basis. IC50 (µg/mL) is the concentration of antioxidant required to scavenge 50% of DPPH present in the mixture. c Trolox equivalents per gram of extract powder on a dry basis. d Standard free astaxanthin (Sigma-Aldrich) in dimethyl sulfoxide solvent. e Crude extracts obtained in different solvents by using one-pot cell wall disruption and extraction at 200 rpm for 30 min. b


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CONCLUSIONS In this study, we proposed a very mild one-pot strategy to effectively achieve the complete recovery of the bioactive natural astaxanthin from the mature cysts of H. pluvialis with high yields of 47 wt%. We developed a simple one-pot method for the simultaneous cell wall disruption and astaxanthin extraction. This strategy could successfully overcome the need of the mandatory pretreatment step for the complete recovery of astaxanthin with high antioxidant activity. A high astaxanthin recovery of >31.6 mg/g dry weight of H. pluvialis was attained by completely rupturing the cell wall of the cyst cells under very mild conditions (200 rpm, room temperature, and atmospheric pressure) and brief extraction (30 min). Astaxanthin recovery was of the highest in ethanol, followed by that in acetone, ethyl acetate, IPA, and hexane. This approach also overcomes the need for the prerequisite drying process of wet microalgae. Extracts obtained using the one-pot method exhibited remarkable antiradical scavenging activity. Alcoholic extracts (IPA and ethanol) showed the highest phenolic contents. The antiradical scavenging activity of hexane and ethyl acetate was the highest, showing good stability of astaxanthin in these solvents. Food-grade solvents can be promising candidates for use in pharmaceutical industries. The operation time and energy required can be further reduced by conducting studies to develop a continuous one-pot extractor and explore its application in biorefineries. To the best of our knowledge, this is the first mild approach that allows the extraction of completely functioning astaxanthin in a short time. This novel one-step mild process performed using food-grade solvents might be useful for continuous operation in biorefineries for sensitive value-added products.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Information about astaxanthin structure, current status of astaxanthin extraction, TGA profiles of dried H. pluvialis, HPLC chromatograms of the extracts, SEM images of H. pluvialis before and after the one-pot extraction, experimental procedure of color determination are available in the Supporting Information (PDF)

ACKNOWLEDGMENTS This research was supported by the Technology Development Program to Solve Climate Changes of the National Research Foundation (NRF) and funded by the Ministry of Science, ICT & Future Planning (2017M1A2A2087635). We appreciate the additional support from the NRF (2018R1D1A1B07047129).

AUTHOR’S CONTRIBUTIONS Prof. Jaehoon Kim (corresponding author) and Prof. Sang Jun Sim conceived the concept of the safe extraction of astaxanthin from Haematococcus pluvialis. Muhammad Irshad (first author) performed experimental studies, analyzed the extracts, and drafted the manuscript. Dr. Min Eui Hong conducted the biotechnical study of Haematococcus pluvialis algae under the supervision of Prof. Sang Jun Sim. Dr. Aye Aye Myint designed the antioxidant activity test. Prof. J. Kim revised the manuscript and supervised the project. All the authors contributed to the discussion and drafting of the manuscript.


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AUTHOR’S DECLARATION This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All the authors have approved the manuscript and agree with submission to your esteemed journal.

CONFLICT OF INTEREST The authors have no affiliation with or no involvement in any organization with financial benefits or non-financial interests in the materials discussed in the manuscript. There are no conflicts of interest to declare. No conflicts, informed consent, and human or animal rights applicable.

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Graphical abstract: The green extraction and sustainability of thermolabile, bioactive astaxanthin is challenging due to presence of resistive cell wall in Haematococcus pluvialis.


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One-pot, simultaneous cell wall disruption and complete extraction of

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