Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure Homogenization for Astaxanthin Recovery

Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure... applied sciences Article Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure Homogenization for Astaxanthin Recovery 1 , 2 3 3 3 4 Ramasamy Praveenkumar , Jiye Lee , Durairaj Vijayan , Soo Youn Lee , Kyubock Lee , 5 5 6 6 , Sang Jun Sim , Min Eui Hong , Young-Eun Kim and You-Kwan Oh * Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark; praveen@ruc.dk Center for Virtual Learning Technologies, Roskilde University, 4000 Roskilde, Denmark Climate Change Research Division, Korea Institute of Energy Research, Daejeon 34129, Korea; jiye.lee@kier.re.kr (J.L.); vijayan.mibi@gmail.com (D.V.); syl@kier.re.kr (S.Y.L.) Graduate School of Energy Science and Technology, Chungnam National University, Daejeon 34134, Korea; kyubock.lee@cnu.ac.kr Department of Chemical & Biological Engineering, Korea University, Seoul 02841, Korea; simsj@korea.ac.kr (S.J.S.); finalarea@naver.com (M.E.H.) School of Chemical & Biomolecular Engineering, Pusan National University, Busan 46241, Korea; alflso0191@pusan.ac.kr * Correspondence: youkwan@pusan.ac.kr; Tel.: +82-51-510-2395 Received: 12 December 2019; Accepted: 6 January 2020; Published: 10 January 2020 Abstract: Haematococcus pluvialis accumulates astaxanthin, which is a high-value antioxidant, during the red cyst stage of its lifecycle. The development of a rigid cell wall in the cysts hinders the recovery of astaxanthin. We investigated morphological changes and cell disruption of mature H. pluvialis cyst cells while using high-pressure homogenization for astaxanthin extraction. When treated with French-press-cell (pressure, 10,000–30,000 psi; passage, 1–3), the intact cyst cells were significantly broken or fully ruptured, releasing cytoplasmic components, thereby facilitating the separation of astaxanthin by ethyl acetate. Fluorescence microscopy observations using three di erent fluorescent dyes revealed that a greater degree of cell breakage caused greater external dispersion of astaxanthin, chlorophyll, lipids, proteins, and carbohydrates. The mechanical treatment resulted in a high cell disruption rate of up to 91% based on microscopic cell typing and Coulter methods. After the ethyl acetate extraction, the astaxanthin concentration significantly increased by 15.2 mg/L in proportion to the increase in cell disruption rate, which indicates that cell disruption is a critical factor for solvent-based astaxanthin recovery. Furthermore, this study recommends a synergistic combination of the fast instrumental particle-volume-distribution analysis and microscope-based morphologic phenotyping for the development of practical H. pluvialis biorefinery processes that co-produce various biological products, including lipids, proteins, carbohydrates, chlorophyll, and astaxanthin. Keywords: Haematococcus pluvialis; high-pressure homogenization; astaxanthin; cyst; cell disruption 1. Introduction Microalgal biomass is currently considered as a promising resource for valuable biochemicals and 0 0 biofuels [1,2]. Especially, astaxanthin (3,3 -dihydroxy- -carotene-4,4 -dione), which is a secondary ketocarotenoid compound, has been widely applied in feed, cosmetic, and pharmaceutical industries, owing to its excellent antioxidant, anti-inflammatory, and anti-cancer properties [3–5]. Although synthetic astaxanthin is extensively used as feed additives in aquaculture, natural astaxanthin is preferred over synthetic form for human consumption in view of its mixture of isomers and safety Appl. Sci. 2020, 10, 513; doi:10.3390/app10020513 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 513 2 of 10 concerns [6,7]. Several commercial-scale facilities currently produce natural astaxanthin from green microalga Haematococcus pluvialis having high astaxanthin content (~4%, w/w) [8,9]. H. pluvialis cell wall is dicult to destroy due to the considerable thickness and rigidity that are associated with its multiple layers formed during the transformation from green vegetative cells to astaxanthin-rich red cysts [10,11]. Apart from astaxanthin recovery, a biorefinery process that can co-produce other cellular components, such as lipids, proteins, and carbohydrates, can significantly reduce the cost of large-scale H. pluvialis-based astaxanthin production [12,13]. Accordingly, e orts to develop an environmental-friendly and cost-e ective cell disruption process from H. pluvialis is practically in progress. Various chemical and supercritical fluid extractions of astaxanthin from H. pluvialis biomass have been extensively studied [14,15]. However, the chemical treatment is potentially harmful with the excessive use of strong organic solvent (s) resulting in a lower stability and quality of astaxanthin [8]. The supercritical CO extraction process requires longer operation time and, generally, the use of co-solvent(s) and/or physicochemical pretreatment. Furthermore, this method is very expensive for large-scale biorefining applications [8,14]. High-pressure homogenization has been proposed as a relatively simple and importantly scalable cell disruption method for industrial-scale microalgal biorefining processes among various mechanical techniques, including bead milling, high-pressure homogenization, ultrafine grinding, autoclave, and sonication [8,12]. However, although there have been several studies on oleaginous Chlorella and Nannochloropsis species, especially for biodiesel production, application on harder aplanospore-forming species (e.g., H. pluvialis) is relatively limited [16]. In this study, the eciency of astaxanthin recovery according to cellular disruption level of H. pluvialis cyst cells was investigated by controlling high pressures (10,000–30,000 psi) and passages (1–3) of a French press cell as a model high-pressure homogenization method. During the mechanical cell disruption, the cell types were classified as intact, leaky, and ruptured based on optical microscopy. The size distribution of cellular particles and spatial distribution of intracellular biomolecules (e.g., astaxanthin, lipids, proteins, carbohydrates, and chlorophyll) were also characterized while using a Coulter counter and staining-based fluorescence microscopy techniques, respectively. Specific insights on their morphological changes and distributions will be beneficial for ecient scale-up and biorefinery applications. Finally, the technical feasibility of a fast instrumental Coulter counter was investigated as an alternative to the conventional time-consuming microscopic cell classification as an indicator of the algal cell disruption level [17]. 2. Materials and Methods 2.1. Materials Professor Sang Jun Sim of Korea University, Seoul, Korea provided the intact cysts of H. pluvialis NIES-144 that were used in this study. NIES-144 strain was originally obtained from the National Institute for Environmental Studies (NIES), Tsukuba, Japan, and cultivated in thin-film photobioreactors (working volume, ~63 L) under outdoor photoautotrophic conditions while using a CO -rich (~3.5%, v/v) power-plant flue gas (Korean District Heating Co., Pangyo, Gyeonggi, Korea). Comprehensive medium composition and culture conditions are available in the literature [18]. The H. pluvialis cysts were harvested by centrifugation (1100 g, 10 min., 4 C; Supra 22K, Hanil Science Inc., Gimpo, Korea), washed three times with purified water (Millipore Milli-Q system, Japan), and then freeze-dried for 48 h (FD5512, IlShin BioBase Co., Gyeonggi, Korea). The lyophilized H. pluvialis biomass was preserved in a sterile vacuum bag and stored at20 C in a dark environment until further use. All of the organic solvents and chemicals used in this study were of analytical grade and from Junsei (Tokyo, Japan) and Sigma Aldrich (St. Louis, MO, USA). Astaxanthin standard was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The fatty acid methyl ester (FAME) (Mix RM3, Mix RM5, GLC50, and GLC70) and sugar (D-glucose, D-arabinose, D-mannose, D-galactose, and D-xylose) standards were supplied from Sigma Aldrich (St. Louis, MO, USA). Appl. Sci. 2020, 10, 513 3 of 10 2.2. Mechanical Cell Disruption High-pressure homogenization method (French press cell, Thermo Electron Co., Waltham, MA, USA) was applied to algal biomass in three passes at three di erent pressures (10,000, 20,000, and 30,000 psi) to disrupt H. pluvialis cysts. The freeze-dried H. pluvialis biomass was dispersed in distilled water (Millipore Milli-Q system, Tokyo, Japan) at 1.0 g/L and vigorously mixed in a vortex (Vortex 3, IKA, Staufen, Germany) to ensure the homogeneity of the algal sample. The maximum internal capacity of the French press cell was 30 mL and the working volume in this study was fixed at 20 mL. In this process, the algal cells (1.0 g/L) are forced to flow through a very small orifice under high-pressure conditions, and, as a result, they could be disrupted by synergistic mechanical e ects, such as cavitation, turbulence, and shear stress [12]. 2.3. Microscopic Observation The cell disruption and distribution of internal biomolecules of H. pluvialis cyst cells were observed under a bright field and fluorescence microscope (Carl Zeiss imager A2, Oberkochen, Germany). In the bright field mode, after the high-pressure homogenization, H. pluvialis cysts containing astaxanthin (red color) could be largely classified into intact, leaky, and ruptured types while considering their morphological changes due to mechanical breakages. Cell staining was carried out in the order of proteins, carbohydrates, and lipids in order to simultaneously visualize the cell wall and various cytoplasmic components. To stain proteins, 100 L of sodium carbonate bu er (0.1 M) and 10 L of fluorescein isothiocyanate solution (FITC, 10 mg/mL) were added into the algal sample. Afterwards, the solution was stirred for 1 h at room temperature. Next, for carbohydrate staining, the cells were washed twice with phosphate bu er saline (pH = 7.2), and 100 L of Calcofluor white reagent (300 mg/L) was added thereto and then incubated for 30 min. Again, washing was performed twice with phosphate bu er saline (pH = 7.2), followed by the addition of 10 L of Nile red solution (20 mg/mL) for neutral lipid detection. Following this, the sample was incubated in a dark environment for 10 min. and then washed twice with phosphate bu er saline (pH = 7.2). In the fluorescent mode, the blue color of the carbohydrates was observed while using filter set 49, the green color of proteins and red color of chlorophyll auto-fluorescence was observed using filter set A9, and the yellow color of neutral lipids was observed using filter set 09. 2.4. Cell Disruption Eciency Estimation Instrumental particle-counting and microscopic cell typing methods evaluated cell disruption TM ecacy during the high-pressure homogenization. A Coulter counter (Multisizer 4, Beckman Coulter, Brea, CA, USA) with a 100 m aperture tube was used for determining the bio-volume, size, and number of particles in the range of 2–60 m. The sample volume was adjusted to 100 mL while using Isoton II electrolyte solution (Beckman Coulter, Brea, CA, USA) in the way that the particle concentration was kept below 10%. The cell disruption eciency (%) was estimated from the total bio-volume reduction degree of the homogenized sample versus the untreated control (100%). The total volume of particles was calculated by summing each volume of all the particles in the selected size range (especially 15–60 m). The cell disruption degree (i.e., the remaining intact type no/total cell no. after the mechanical treatment) was also estimated by manually sorting over 100 cells while using an improved Neubauer counting chamber (C-Chip, DHC-N01, iNCYTO, Chungnam, Korea) based on the optical microscope mentioned above. 2.5. Other Analytical Methods For astaxanthin extraction, 1 mL of French-press-treated H. pluvialis cells was vigorously mixed in a vortex mixer (Vortex 3, IKA, Germany) with 5 mL of ethyl acetate for 10 min. at room temperature. The mixture was separated by centrifugation (2000 g, 10 min.; Combi-514R, Hanil Science Inc., Gimpo, Korea) and an astaxanthin-containing organic fraction was collected and evaporated (N-EVAP Appl. Sci. 2020, 10, 513 4 of 10 evaporator; Organomation Associates Inc., Berlin, MA, USA). After saponification reaction with 0.025 N NaOH, free (de-esterified) astaxanthin content was analyzed while using a high-performance liquid chromatography (HPLC, Agilent 1260 series, Agilent, Santa Clara, CA, USA) that was equipped with a variable wavelength detector (VWD) and 250 4.6 mm YMC carotenoid column (Tokyo, Japan). The detailed extraction and HPLC conditions are detailed in the literature [6,19]. Lipid content of the freeze-dried H. pluvialis cysts was estimated by the FAME amount, according to a direct transesterification method, followed by gas chromatography (GC) [6,19]. Briefly, for lipid extraction, 2 mL of the chloroform/methanol (2:1, v/v) was added to the algal cells (~10 mg in a 12 mL Pyrex-glass tube). For the FAME analysis, subsequently, 1 mL of chloroform containing heptadecanoic acid (C17:0) as an internal standard (500 g/L), 1 mL of methanol, and 300 L of H SO (95%) were 2 4 added to the glass tube. The tube was incubated at 100 C for 10 min. (HS R200 heating-block reactor, Humas, Daejeon, Korea), cooled to room temperature, and then supplemented with 1 mL of distilled water. Finally, the FAME-containing organic phase was filtered by a PVDF (polyvinyl difluoride) syringe filter and then analyzed by GC that was equipped with a flame-ionization detector and a 0.32 mm (ID) 30 m HP-INNOWax capillary column (Agilent Technologies, Santa Clara, California, USA). The detailed GC conditions are available in [19]. Carbohydrate amount was estimated according to the NREL/TP-510-42618 protocol [20]. For cell hydrolysis, approximately 300 mg of the lyophilized H. pluvialis cells was mixed with 3 mL of H SO solution (72%) in a Pyrex-glass tube and then incubated for 60 min. at 30 C. Subsequently, 2 4 84 mL of purified water (Millipore Milli-Q system, Tokyo, Japan) was added and further incubated for 1 h at 121 C while using an autoclave (Napco 8000-DSE, Thermo Scientific Co., Waltham, MA, USA). The carbohydrate extract was cooled down to room temperature, centrifuged at 15,000 g for 5 min. (Combi-514R, Hanil Science Inc., Korea), and then PVDF syringe-filtered. Finally, the filtrate was analyzed using a HPLC equipped with a refractive-index (RI) detector (Agilent 1260, Agilent Co., Santa Clara, CA, USA) and Aminex HPX-87H column (300 7.8 mm, Bio-Rad, Hercules, CA, USA). As an eluent, 5 mM H SO solution was used at a flow rate of 0.5 mL/min. The temperatures of column 2 4 and RI detector were set at 60 C and 50 C, respectively. 3. Results and Discussion 3.1. Morphological Changes by Mechanical Disruption The e ect of French press cell treatment (10,000 psi, 1 pass) on mature H. pluvialis cysts were evaluated morpho-dynamically while using a combination of light and fluorescence microscopy (Figure 1). In the phase-contrast mode of optical microscope, the intact H. pluvialis cysts showed significant amounts of astaxanthin (bright red) and chlorophyll (green) inside the cells. The high-pressure homogenization could partially damage or completely rupture the cell walls of the cysts, resulting in significant releases of the cytoplasmic constituents into the surrounding medium. The H. pluvialis cysts could be largely categorized into intact, leaky, and ruptured types, depending on their degree of destruction (see Figure 2 for their distribution). Three di erent fluorescent probes of Calcofluor white, Nile red, and FITC were used under fluorescence microscopy, respectively, in order to simultaneously visualize the cell wall and cytoplasmic polysaccharides (blue), neutral lipids (yellow), and proteins (green) of H. pluvialis cyst cells. Figure 1 shows astaxanthin (magenta) and chlorophyll (red) auto-fluoresce without chemical staining. The total carbohydrate content of the H. pluvialis cysts that were incubated under the outdoor conditions while using a CO -rich (~3.5%, v/v) power-plant flue gas was estimated to be 12% (w/w). With the Calcofluor white dye, the polysaccharide fractions of the cell wall and the cytoplasmic contents were stained blue under the DAPI filter. The intact encysted cells showed a continuous blue color on their cell walls, because the H. pluvialis cell wall is mostly composed of mannan and cellulose polysaccharides [21,22]. However, significant dispersion of the blue-colored cell wall and cytoplasmic polysaccharides were observed after mechanical disruption. Appl. Sci. 2020, 10, 513 5 of 10 Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 10 Nile-red-stained neutral lipids are usually detected as cytoplasmic yellow-colored droplets in microscopy. The total lipid content of the H. pluvialis cyst cell was as high as 40% (w/w) when oleaginous microalgal species, such as Chlorella [23] and Nannochloropsis [24], under fluorescent estimated in terms of FAME amount by GC. This is similar to the typical lipid compositions of H. microscopy. The total lipid content of the H. pluvialis cyst cell was as high as 40% (w/w) when estimated pluvialis (32–37% (w/w)) harvested from the red cultivation stage [25]. However, it should be noted in terms of FAME amount by GC. This is similar to the typical lipid compositions of H. pluvialis (32–37% that, in H. pluvialis cysts, astaxanthin biomolecules co-exist inside the lipid body mostly in mono- and (w/w)) harvested from the red cultivation stage [25]. However, it should be noted that, in H. pluvialis di-ester forms with long-chain fatty acids [26,27]. Therefore, under the present fluorescence cysts, astaxanthin biomolecules co-exist inside the lipid body mostly in mono- and di-ester forms with microscopy condition, the former (magenta) and latter (yellow) cannot be clearly distinguished from long-chain fatty acids [26,27]. Therefore, under the present fluorescence microscopy condition, the the intact and leaky cell types. However, interestingly, after almost the complete rupture of the H. former (magenta) and latter (yellow) cannot be clearly distinguished from the intact and leaky cell pluvialis cyst cells, several yellow-colored lipid droplets appeared from the dispersed cytoplasm. The types. However, interestingly, after almost the complete rupture of the H. pluvialis cyst cells, several protein fractions from H. pluvialis cells fluoresced green after the FITC staining. As expected, the yellow-colored lipid droplets appeared from the dispersed cytoplasm. The protein fractions from H. intact cysts showed a relatively uniform green distribution in the cytosol, except for lipids, pluvialis cells fluoresced green after the FITC staining. As expected, the intact cysts showed a relatively astaxanthin, and chlorophyll. However, following the mechanical treatment, the intensity of gree n uniform green distribution in the cytosol, except for lipids, astaxanthin, and chlorophyll. However, color was significantly reduced. This might be mainly attributed to the significant loss of water- following the mechanical treatment, the intensity of green color was significantly reduced. This might soluble proteins in the medium. Overall, the degree of dispersion of the internal constituents, be mainly attributed to the significant loss of water-soluble proteins in the medium. Overall, the degree including astaxanthin, increased in proportion to the degree of cell rupture. This resulted in the of dispersion of the internal constituents, including astaxanthin, increased in proportion to the degree effective recovery of astaxanthin from the robust H. pluvialis cysts by subsequent solvent extraction of cell rupture. This resulted in the e ective recovery of astaxanthin from the robust H. pluvialis cysts (see next sections). by subsequent solvent extraction (see next sections). Figure 1. Microscopic images of H. pluvialis cysts after the high-pressure homogenization treatment (10,000 psi and one pass). Cell shapes were classified into three types (i.e., intact, leaky, and ruptured) Figure 1. Microscopic images of H. pluvialis cysts after the high-pressure homogenization treatment considering their morphological changes by the mechanical breakages. See colored arrows for each cell (10,000 psi and one pass). Cell shapes were classified into three types (i.e., intact, leaky, and ruptured) type. From left to right, optical phase-contrast images; fluorescent images under DAPI filter showing considering their morphological changes by the mechanical breakages. See colored arrows for each the cell wall and other polysaccharides (blue stained with Calcofluor white); fluorescent images under cell type. From left to right, optical phase-contrast images; fluorescent images under DAPI filter fluorescein isothiocyanate solution (FITC) long-pass filter showing the proteins (green stained with showing the cell wall and other polysaccharides (blue stained with Calcofluor white); fluorescent FITC), lipids (yellow stained with Nile red), chlorophyll (red by auto-fluorescence), and astaxanthin images under fluorescein isothiocyanate solution (FITC) long-pass filter showing the proteins (green (magenta by auto-fluorescence). stained with FITC), lipids (yellow stained with Nile red), chlorophyll (red by auto-fluorescence), and astaxanthin (magenta by auto-fluorescence). Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 10 3.2. Cell Type Distribution by Microscopic Counting Figure 2 shows the relative cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure intensity, 10,000–30,000 psi; pass, 1–3 times). The morphological cell types were calculated by sorting over 100 cells while using the improved Neubauer counting chamber under microscopic observation (also see Figure 1). After the homogenization in one pass at a pressure of 10,000 psi, the number of intact cyst cells accounted for ca. 45%, whereas the rest of the initial cysts became either leaky or ruptured completely (cell disruption, 54.8 ± 0.3%). Here, the cell disruption (%) was estimated by subtracting the % ratio of both the leaky and ruptured cells from the initial cells (100%). The cell disruption degree significantly increased to 80.2 ± 6.0% and 89 ± 0.8%, respectively, when the algal-suspension was homogenized once and twice more at the same pressure. On the contrary, at higher pressures of 20,000–30,000 psi, the cell disruption efficiency of H. pluvialis cysts did not improve when compared to that of the 10,000 psi treatment even if the passage number was increased from 1 to 3. The cell disruption efficacy of the high-pressure homogenization process can be influenced by the algal cell concentration, as well as the homogenizer specification (loading pressure and number of passage) [12]. Lowering the cell dosage might be considered in order to improve the cell disruption rate to more than 90%. However, it should be noted that this approach is not generally recommended in terms of large-scale biorefining of H. pluvialis biomass on an industrial scale [8]. Although the high-pressure homogenization has been recommended as a relatively efficient and scalable method among various physicochemical cell disruption techniques for large-scale algal biorefining [8,12], studies on H. pluvialis cyst cells are very limited. Praveenkumar et al. [19] reported that, when compared to ionic liquid-based extractions, the 30,000 psi-fixed high-pressure homogenization resulted in a high yield of 24 pg astaxanthin/cell from wet H. pluvialis cysts that were cultured under small-scale laboratory conditions. However, the cell disruption rate and key operating parameters (especially pressure intensity and number of passages) were not elucidated. Safi et al. [17] obtained the highest protein extraction yield (40%) from lyophilized H. pluvialis biomass while using a high-pressure cell disruptor that was fixed at 2700 bar (39,150 psi), as compared to manual grinding, alkaline treatment, and ultrasonication treatments. Moreover, they reported that, under the same condition, the maximal protein recovery efficiency showed a decreasing trend in accordance with the increasing order of cell wall rigidity among the tested algal strains: Arthrospira platensis < Porphyridium cruentum < Chlorella vulgaris < Nannochloropsis oculata < H. pluvialis. Detailed investigation into cell physiology, cell wall biology, and physical strength of H. pluvialis cells is required in order to develop an economical high-pressure homogenization process. In fact, this insight would create high potentialities for a synergistic or novel process that utilizes various existing biological–physicochemical Appl. Sci. 2020, 10, 513 6 of 10 treatments. Figure 2. Cell type distribution of H. pluvialis cysts after various high-pressure homogenization Figure 2. Cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure, 10,000–30,000 psi; passage, 1–3). The cell types were classified into intact, leaky, treatments (pressure, 10,000–30,000 psi; passage, 1–3). The cell types were classified into intact, leaky, and ruptured according to the morphological features that are based on the microscopic observation (Figure 1). 3.2. Cell Type Distribution by Microscopic Counting Figure 2 shows the relative cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure intensity, 10,000–30,000 psi; pass, 1–3 times). The morphological cell types were calculated by sorting over 100 cells while using the improved Neubauer counting chamber under microscopic observation (also see Figure 1). After the homogenization in one pass at a pressure of 10,000 psi, the number of intact cyst cells accounted for ca. 45%, whereas the rest of the initial cysts became either leaky or ruptured completely (cell disruption, 54.8 0.3%). Here, the cell disruption (%) was estimated by subtracting the % ratio of both the leaky and ruptured cells from the initial cells (100%). The cell disruption degree significantly increased to 80.2  6.0% and 89  0.8%, respectively, when the algal-suspension was homogenized once and twice more at the same pressure. On the contrary, at higher pressures of 20,000–30,000 psi, the cell disruption eciency of H. pluvialis cysts did not improve when compared to that of the 10,000 psi treatment even if the passage number was increased from 1 to 3. The cell disruption ecacy of the high-pressure homogenization process can be influenced by the algal cell concentration, as well as the homogenizer specification (loading pressure and number of passage) [12]. Lowering the cell dosage might be considered in order to improve the cell disruption rate to more than 90%. However, it should be noted that this approach is not generally recommended in terms of large-scale biorefining of H. pluvialis biomass on an industrial scale [8]. Although the high-pressure homogenization has been recommended as a relatively ecient and scalable method among various physicochemical cell disruption techniques for large-scale algal biorefining [8,12], studies on H. pluvialis cyst cells are very limited. Praveenkumar et al. [19] reported that, when compared to ionic liquid-based extractions, the 30,000 psi-fixed high-pressure homogenization resulted in a high yield of 24 pg astaxanthin/cell from wet H. pluvialis cysts that were cultured under small-scale laboratory conditions. However, the cell disruption rate and key operating parameters (especially pressure intensity and number of passages) were not elucidated. Safi et al. [17] obtained the highest protein extraction yield (40%) from lyophilized H. pluvialis biomass while using a high-pressure cell disruptor that was fixed at 2700 bar (39,150 psi), as compared to manual grinding, alkaline treatment, and ultrasonication treatments. Moreover, they reported that, under the same condition, the maximal protein recovery eciency showed a decreasing trend in accordance with the increasing order of cell wall rigidity among the tested algal strains: Arthrospira platensis < Porphyridium cruentum < Chlorella vulgaris < Nannochloropsis oculata < H. pluvialis. Detailed investigation into cell physiology, cell wall biology, and physical strength of H. pluvialis cells is required in order to develop an Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 10 Appl. Sci. 2020, 10, 513 7 of 10 and ruptured according to the morphological features that are based on the microscopic observation (Figure 1). economical high-pressure homogenization process. In fact, this insight would create high potentialities for a synergistic or novel process that utilizes various existing biological–physicochemical treatments. 3.3. Bio-Particle Distribution by Coulter Counter and Astaxanthin Recovery 3.3. Bio-Particle Distribution by Coulter Counter and Astaxanthin Recovery Figure 3 shows the bio-volume distribution and total bio-volume of H. pluvialis bio-particles before and after French press cell treatment with different pressures (10,000–30,000 psi) and then Figure 3 shows the bio-volume distribution and total bio-volume of H. pluvialis bio-particles before passes (1–3). Here, “bio-particles” collectively refer to the ruptured cells, cell debris, and internal and after French press cell treatment with di erent pressures (10,000–30,000 psi) and then passes (1–3). biomolecules that have leaked outwards, as well as the initial intact cells. In the untreated control, Here, “bio-particles” collectively refer to the ruptured cells, cell debris, and internal biomolecules that most of the H. pluvialis cysts were of 15–60 µ m cell size and they exhibited a relatively large particle have leaked outwards, as well as the initial intact cells. In the untreated control, most of the H. pluvialis 7 3 volume distribution (~4.1 × 10 µm /mL) (Figure 3a). However, when homogenized at a pressure of cysts were of 15–60 m cell size and they exhibited a relatively large particle volume distribution 10,000 psi, the particle volumes of bio-particles in the same range significantly decreased in 7 3 (~4.1 10 m /mL) (Figure 3a). However, when homogenized at a pressure of 10,000 psi, the particle 7 3 7 3 proportion to the increase in the number of passage: ~1.7 × 10 µm /mL for one pass, ~1.3 × 10 µm /mL volumes of bio-particles in the same range significantly decreased in proportion to the increase in 7 3 for two passes, and ~0.5 × 10 µm /mL for three passes. On the contrary, the volumes of the bio- 7 3 7 3 the number of passage: ~1.7  10 m /mL for one pass, ~1.3  10 m /mL for two passes, and particles smaller than ~15 µ m increased slightly when compared to the untreated control. These 7 3 ~0.5  10 m /mL for three passes. On the contrary, the volumes of the bio-particles smaller than changes were induced by the mechanical destruction of the initial cyst cells (see Figure 1 for ~15 m increased slightly when compared to the untreated control. These changes were induced by morphological changes). the mechanical destruction of the initial cyst cells (see Figure 1 for morphological changes). Figure 3. Changes in volume distribution (a) and total volume (b) of H. pluvialis bio-particles after Figure 3. Changes in volume distribution (a) and total volume (b) of H. pluvialis bio-particles after mechanical disruptions with di erent pressures and passes. The bio-particles refer to all internal mechanical disruptions with different pressures and passes. The bio-particles refer to all internal components as well as intact, leaky, and ruptured cyst cells. Representative particle-size-volume components as well as intact, leaky, and ruptured cyst cells. Representative particle-size-volume distributions after the 10,000 psi-homogenizations at three di erent passes were presented based on distributions after the 10,000 psi-homogenizations at three different passes were presented based on the Coulter counter analysis. Total volumes were calculated by summing each volume of all particles the Coulter counter analysis. Total volumes were calculated by summing each volume of all particles sized between 15 and 60 m. sized between 15 and 60 µ m. Figure 3b plotted the changes in the total bio-volumes of the bio-particles after the homogenization Figure 3b plotted the changes in the total bio-volumes of the bio-particles after the treatments. The total bio-volume of the homogenized bio-particles was calculated by summing homogenization treatments. The total bio-volume of the homogenized bio-particles was calculated the volume of each bio-particle between 15 to 60 m while considering their characteristic volume by summing the volume of each bio-particle between 15 to 60 µ m while considering their 9 3 reductions. The total bio-volume of the untreated H. pluvialis cysts (1.9  10 m /mL) decreased characteristic volume reductions. The total bio-volume of the untreated H. pluvialis cysts (1.9 × 10 almost linearly with an increasing number of passes (1–3), irrespective of the tested pressure intensity m /mL) decreased almost linearly with an increasing number of passes (1–3), irrespective of the tested (10,000–30,000 psi). However, no significant positive e ect of the high pressures (20,000–30,000 psi) was pressure intensity (10,000–30,000 psi). However, no significant positive effect of the high pressures observed as compared to the case of 10,000 psi, which is similar to that of the microscopic cell typing (20,000–30,000 psi) was observed as compared to the case of 10,000 psi, which is similar to that of the (Figure 2). The cell disruption eciency could be simply derived from the degree of total volume microscopic cell typing (Figure 2). The cell disruption efficiency could be simply derived from the reduction of the homogenized samples (versus the untreated control, 100%), and its maximal value degree of total volume reduction of the homogenized samples (versus the untreated control, 100%), was estimated to be 91.8 2.2%. This value was also almost equal to the highest value (89 0.8%) that and its maximal value was estimated to be 91.8 ± 2.2%. This value was also almost equal to the highest was obtained from the microscopic cell counting in Figure 2. value (89 ± 0.8%) that was obtained from the microscopic cell counting in Figure 2. When comparing the cell disruption eciencies that were calculated from Coulter counting and When comparing the cell disruption efficiencies that were calculated from Coulter counting and microscopic cell typing methods, a statistically good correlation (r = 0.95) was obtained (Figure 4a). microscopic cell typing methods, a statistically good correlation (r = 0.95) was obtained (Figure 4a). Furthermore, by the subsequent ethyl acetate extraction, astaxanthin was recovered from the H. pluvialis Furthermore, by the subsequent ethyl acetate extraction, astaxanthin was recovered from the H. biomass treated under various high-pressure homogenization conditions (see Figures 2 and 3). As pluvialis biomass treated under various high-pressure homogenization conditions (see Figures 2 and Appl. Sci. 2020, 10, 513 8 of 10 Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 10 3). As expected, the astaxanthin concentration increased almost linearly from 5.3 to 15.2 mg/L in expected, the astaxanthin concentration increased almost linearly from 5.3 to 15.2 mg/L in proportion proportion to the increase in cell disruption level (Figure 4b), which indicated that cell disruption is to the increase in cell disruption level (Figure 4b), which indicated that cell disruption is a critical a critical factor for solvent-based astaxanthin recovery. The astaxanthin content of the H. pluvialis factor for solvent-based astaxanthin recovery. The astaxanthin content of the H. pluvialis biomass was biomass was estimated to be 1.1% (w/w). Astaxanthin from the freeze-dried H. pluvialis cyst cells estimated to be 1.1% (w/w). Astaxanthin from the freeze-dried H. pluvialis cyst cells could also be could also be extracted while using solely ethyl acetate, although its concentration was as low as 5.3 extracted while using solely ethyl acetate, although its concentration was as low as 5.3 mg/L. The mg/L. The statistical r values for relationships between the astaxanthin recovery and cell disruption statistical r values for relationships between the astaxanthin recovery and cell disruption degree degree were estimated to be 0.83 and 0.86 for the microscopic and Coulter counter methods, were estimated to be 0.83 and 0.86 for the microscopic and Coulter counter methods, respectively. respectively. This result suggests that the Coulter counter method can be used effectively for the rapid This result suggests that the Coulter counter method can be used e ectively for the rapid assessment assessment of H. pluvialis cell disruption for astaxanthin recovery. Microscopy phenotyping generally of H. pluvialis cell disruption for astaxanthin recovery. Microscopy phenotyping generally requires requires time-consuming and laborious experimentation by skilled personnel. However, it should be time-consuming and laborious experimentation by skilled personnel. However, it should be noted that noted that this approach is very useful in understanding the actual morphological changes of the this approach is very useful in understanding the actual morphological changes of the rigid H. pluvialis rigid H. pluvialis cysts and the spatial distribution of the target biomolecules during mechanical cysts and the spatial distribution of the target biomolecules during mechanical treatment (Figure 1). treatment (Figure 1). Therefore, a synergistic combination of these methods is recommended for the Therefore, a synergistic combination of these methods is recommended for the development of practical development of practical H. pluvialis biorefinery processes that co-produce various bio-products, H. pluvialis biorefinery processes that co-produce various bio-products, including lipids, proteins, including lipids, proteins, carbohydrates, chlorophyll, and astaxanthin. Several analytical techniques carbohydrates, chlorophyll, and astaxanthin. Several analytical techniques have been reported in have been reported in order to evaluate the disintegration efficiency of algal biomass by mechanical order to evaluate the disintegration eciency of algal biomass by mechanical treatments: particle-size treatments: particle-size analyzer (homogenization for Chlamydomonas reinhardtii and analyzer (homogenization for Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata [28]; bead Pseudokirchneriella subcapitata [28]; bead beating for C. reinhardtii [29]), dynamic light scattering beating for C. reinhardtii [29]), dynamic light scattering (ultrasonication for Parachlorella kessleri [30]), (ultrasonication for Parachlorella kessleri [30]), and flow cytometer (bead milling for Chlorella species and flow cytometer (bead milling for Chlorella species [31,32]). However, after cell disruption, the [31,32]). However, after cell disruption, the morphological characteristics of the algal cell and/or the morphological characteristics of the algal cell and/or the internal target substance(s) have not been internal target substance(s) have not been studied in detail. studied in detail. Figure 4. (a) Cell disruption eciency relationship between microscopic cell typing and instrumental Figure 4. (a) Cell disruption efficiency relationship between microscopic cell typing and instrumental particle-size-distribution measurements (r = 0.95); (b) astaxanthin recovery, depending on cell particle-size-distribution measurements (r = 0.95); (b) astaxanthin recovery, depending on cell 2 2 disruption eciencies (microscope, r = 0.83; Coulter counter, r = 0.86). 2 2 disruption efficiencies (microscope, r = 0.83; Coulter counter, r = 0.86). 4. Conclusions 4. Conclusions High-pressure homogenization might be used for highly ecient cell disruption and astaxanthin High-pressure homogenization might be used for highly efficient cell disruption and recovery from mature H. pluvialis cyst cells, followed by ethyl acetate extraction. French press cell astaxanthin recovery from mature H. pluvialis cyst cells, followed by ethyl acetate extraction. French treatment e ectively destroys the H. pluvialis cyst cells and results in a high disruption rate of up to 91%. press cell treatment effectively destroys the H. pluvialis cyst cells and results in a high disruption rate The homogenized cells can be classified into intact, leaky, and completely ruptured, according to their of up to 91%. The homogenized cells can be classified into intact, leaky, and completely ruptured , morphological changes under an optical microscope. The degree of external dispersion of astaxanthin, according to their morphological changes under an optical microscope. The degree of external lipids, proteins, and carbohydrates was almost proportional to the degree of cell rupture when analyzed dispersion of astaxanthin, lipids, proteins, and carbohydrates was almost proportional to the degree by fluorescence microscopy with specific fluorescent probes. Pressure intensity (10,000–30,000 psi) did of cell rupture when analyzed by fluorescence microscopy with specific fluorescent probes. Pressure not a ect the cell disruption eciency, but an increasing the number of passages (1–3 times) could intensity (10,000–30,000 psi) did not affect the cell disruption efficiency, but an increasing the number significantly improve the cell disruption eciency. After the subsequent ethyl acetate extractions, of passages (1–3 times) could significantly improve the cell disruption efficiency. After the the astaxanthin concentration increased linearly from 5.3 to 15.2 mg/L with increased cell disruption subsequent ethyl acetate extractions, the astaxanthin concentration increased linearly from 5.3 to 15.2 mg/L with increased cell disruption efficacy owing to homogenization optimization. The maximum astaxanthin recovery was estimated to be 1.1% (weight of dry cells). Appl. Sci. 2020, 10, 513 9 of 10 ecacy owing to homogenization optimization. The maximum astaxanthin recovery was estimated to be 1.1% (weight of dry cells). Author Contributions: Conceptualization, R.P. and K.L.; methodology, R.P., J.L., D.V., M.E.H. and S.J.S.; Writing—Original draft preparation, R.P.; Writing—Review and editing, R.P., Y.-K.O., Y.-E.K. and S.Y.L.; supervision, Y.-K.O.; funding acquisition, Y.-K.O. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Research Foundation of Korea (grant number: NRF-2019R1A2C1003463) funded by the Ministry of Science and ICT and the Research/Development Program of the Korea Institute of Energy Research (grant number: KIER-B9-2442-04). Sim, S.J., also would like to acknowledge the support of the Korea CCS R & D Center (Korea CCS 2020 Project) funded by the Ministry of Science and ICT in 2017 (grant number: KCRC-2014M1A8A1049278). Conflicts of Interest: The authors declare no conflict of interest. References 1. Bauer, A.; Minceva, M. Direct extraction of astaxanthin from the microalgae: Haematococcus pluvialis using liquid-liquid chromatography. RSC Adv. 2019, 9, 22779–22789. [CrossRef] 2. Matter, I.A.; Hoang Bui, V.K.; Jung, M.; Seo, J.Y.; Kim, Y.E.; Lee, Y.C.; Oh, Y.K. Flocculation harvesting techniques for microalgae: A review. Appl. Sci. 2019, 9, 3069. [CrossRef] 3. Focsan, A.L.; Polyakov, N.E.; Kispert, L.D. Photo protection of Haematococcus pluvialis algae by astaxanthin: Unique properties of astaxanthin deduced by EPR, optical and electrochemical studies. Antioxidants 2017, 6, 80. [CrossRef] [PubMed] 4. Guerin, M.; Huntley, M.E.; Olaizola, M. Haematococcus astaxanthin: Applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210–216. [CrossRef] 5. Pérez-López, P.; González-García, S.; Je ryes, C.; Agathos, S.N.; McHugh, E.; Walsh, D.; Murray, P.; Moane, S.; Feijoo, G.; Moreira, M.T. Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: From lab to pilot scale. J. Clean. Prod. 2014, 64, 332–344. [CrossRef] 6. Choi, S.A.; Oh, Y.K.; Lee, J.; Sim, S.J.; Hong, M.E.; Park, J.Y.; Kim, M.S.; Kim, S.W.; Lee, J.S. High-eciency cell disruption and astaxanthin recovery from Haematococcus pluvialis cyst cells using room-temperature imidazolium-based ionic liquid/water mixtures. Bioresour. Technol. 2019, 274, 120–126. [CrossRef] [PubMed] 7. Samorì, C.; Pezzolesi, L.; Galletti, P.; Semeraro, M.; Tagliavini, E. Extraction and milking of astaxanthin from: Haematococcus pluvialis cultures. Green Chem. 2019, 21, 3621–3628. [CrossRef] 8. Kim, D.Y.; Vijayan, D.; Praveenkumar, R.; Han, J.I.; Lee, K.; Park, J.Y.; 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. [CrossRef] 9. 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. [CrossRef] 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. [CrossRef] 11. Machado, F.R.S.; Trevisol, T.C.; Boschetto, D.L.; Burkert, J.F.M.; Ferreira, S.R.S.; Oliveira, J.V.; Burkert, C.A.V. Technological process for cell disruption, extraction and encapsulation of astaxanthin from Haematococcus pluvialis. J. Biotechnol. 2016, 218, 108–114. [CrossRef] [PubMed] 12. Lee, S.Y.; Cho, J.M.; Chang, Y.K.; Oh, Y.K. Cell disruption and lipid extraction for microalgal biorefineries: A review. Bioresour. Technol. 2017, 244, 1317–1328. [CrossRef] [PubMed] 13. Khoo, K.S.; Lee, S.Y.; Ooi, C.W.; Fu, X.; Miao, X.; Ling, T.C.; Show, P.L. Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresour. Technol. 2019, 121606. [CrossRef] [PubMed] 14. Reyes, F.A.; Mendiola, J.A.; Ibañez, E.; Del Valle, J.M. Astaxanthin extraction from Haematococcus pluvialis using CO -expanded ethanol. J. Supercrit. Fluids 2014, 92, 75–83. [CrossRef] 15. Sarada, R.; Vidhyavathi, R.; Usha, D.; Ravishankar, G.A. An ecient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J. Agric. Food Chem. 2006, 54, 7585–7588. [CrossRef] 16. Park, J.Y.; Oh, Y.K.; Choi, S.A.; Kim, M.C. Recovery of astaxanthin-containing oil from Haematococcus pluvialis by nano-dispersion and oil partitioning. Appl. Biochem. Biotechnol. 2020, 1–15. [CrossRef] Appl. Sci. 2020, 10, 513 10 of 10 17. Safi, C.; Ursu, A.V.; Laroche, C.; Zebib, B.; Merah, O.; Pontalier, P.Y.; Vaca-Garcia, C. Aqueous extraction of proteins from microalgae: E ect of di erent cell disruption methods. Algal Res. 2014, 3, 61–65. [CrossRef] 18. Choi, Y.Y.; Hong, M.E.; Jin, E.S.; Woo, H.M.; Sim, S.J. Improvement in modular scalability of polymeric thin-film photobioreactor for autotrophic culturing of Haematococcus pluvialis using industrial flue gas. Bioresour. Technol. 2018, 249, 519–526. [CrossRef] 19. Praveenkumar, R.; Lee, K.; Lee, J.; Oh, Y.K. Breaking dormancy: An energy-ecient means of recovering astaxanthin from microalgae. Green Chem. 2015, 17, 1226–1234. [CrossRef] 20. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. NREL/TP-510-42618 analytical procedure—Determination of structural carbohydrates and lignin in biomass. Lab. Anal. Proced. 2008, 1617, 1–16. 21. Hagen, C.; Siegmund, S.; Braune, W.; Botanik, A.; Jena, F.; Planetarium, A. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur. J. Phycol. 2002, 37, 217–226. [CrossRef] 22. Damiani, M.C.; Leonardi, P.I.; Pieroni, O.I.; Cáceres, E.J. Ultrastructure of the cyst wall of Haematococcus pluvialis (Chlorophyceae): Wall development and behaviour during cyst germination. Phycologia 2006, 45, 616–623. [CrossRef] 23. Lee, Y.C.; Lee, H.U.; Lee, K.; Kim, B.; Lee, S.Y.; Choi, M.H.; Farooq, W.; Choi, J.S.; Park, J.Y.; Lee, J.; et al. Aminoclay-conjugated TiO synthesis for simultaneous harvesting and wet-disruption of oleaginous Chlorella sp. Chem. Eng. J. 2014, 245, 143–149. [CrossRef] 24. Wei, L.; Huang, X. Long-duration e ect of multi-factor stresses on the cellular biochemistry, oil-yielding performance and morphology of Nannochloropsis oculata. PLoS ONE 2017, 12, e0174646. [CrossRef] [PubMed] 25. Shah, M.M.R.; Liang, Y.; Cheng, J.J.; Daroch, M. Astaxanthin-producing green microalga Haematococcus pluvialis: From single cell to high value commercial products. Front. Plant Sci. 2016, 7, 531. [CrossRef] 26. Ding, W.; Li, Q.; Han, B.; Zhao, Y.; Geng, S.; Ning, D. Comparative physiological and metabolomic analyses of the hyper-accumulation of astaxanthin and lipids in Haematococcus pluvialis upon treatment with butylated hydroxyanisole. Bioresour. Technol. 2019, 292, 122002. [CrossRef] [PubMed] 27. Peled, E.; Leu, S.; Zarka, A.; Weiss, M.; Pick, U.; Khozin-Goldberg, I.; Boussiba, S. Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (chlorophyceae). Lipids 2011, 46, 851–861. [CrossRef] 28. Lavoie, M.; Bernier, J.; Fortin, C.; Campbell, P.G.C. Cell homogenization and subcellular fractionation in two phytoplanktonic algae: Implications for the assessment of metal subcellular distributions. Limnol. Oceanogr. Methods 2009, 7, 277–286. [CrossRef] 29. Lam, G.P.; Van Der Kolk, J.A.; Chordia, A.; Vermuë, M.H.; Olivieri, G.; Eppink, M.H.M.; Wij els, R.H. Mild and selective protein release of cell wall deficient microalgae with pulsed electric field. ACS Sustain. Chem. Eng. 2017, 5, 6046–6053. [CrossRef] 30. Piasecka, A.; Ciesla, ´ J.; Koczanska, ´ M.; Krzeminska, ´ I. E ectiveness of Parachlorella kessleri cell disruption evaluated with the use of laser light scattering methods. J. Appl. Phycol. 2019, 31, 97–107. [CrossRef] 31. Postma, P.R.; Miron, T.L.; Olivieri, G.; Barbosa, M.J.; Wij els, R.H.; Eppink, M.H.M. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. 2015, 184, 297–304. [CrossRef] [PubMed] 32. Günerken, E.; D’Hondt, E.; Eppink, M.; Elst, K.; Wij els, R. Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling. Algal Res. 2017, 25, 25–31. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Sciences Multidisciplinary Digital Publishing Institute

Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure Homogenization for Astaxanthin Recovery

Loading next page...
 
/lp/multidisciplinary-digital-publishing-institute/morphological-change-and-cell-disruption-of-haematococcus-pluvialis-L2FvzCQJyM
Publisher
Multidisciplinary Digital Publishing Institute
Copyright
© 1996-2020 MDPI (Basel, Switzerland) unless otherwise stated Disclaimer The statements, opinions and data contained in the journals are solely those of the individual authors and contributors and not of the publisher and the editor(s). Terms and Conditions Privacy Policy
ISSN
2076-3417
DOI
10.3390/app10020513
Publisher site
See Article on Publisher Site

Abstract

applied sciences Article Morphological Change and Cell Disruption of Haematococcus pluvialis Cyst during High-Pressure Homogenization for Astaxanthin Recovery 1 , 2 3 3 3 4 Ramasamy Praveenkumar , Jiye Lee , Durairaj Vijayan , Soo Youn Lee , Kyubock Lee , 5 5 6 6 , Sang Jun Sim , Min Eui Hong , Young-Eun Kim and You-Kwan Oh * Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark; praveen@ruc.dk Center for Virtual Learning Technologies, Roskilde University, 4000 Roskilde, Denmark Climate Change Research Division, Korea Institute of Energy Research, Daejeon 34129, Korea; jiye.lee@kier.re.kr (J.L.); vijayan.mibi@gmail.com (D.V.); syl@kier.re.kr (S.Y.L.) Graduate School of Energy Science and Technology, Chungnam National University, Daejeon 34134, Korea; kyubock.lee@cnu.ac.kr Department of Chemical & Biological Engineering, Korea University, Seoul 02841, Korea; simsj@korea.ac.kr (S.J.S.); finalarea@naver.com (M.E.H.) School of Chemical & Biomolecular Engineering, Pusan National University, Busan 46241, Korea; alflso0191@pusan.ac.kr * Correspondence: youkwan@pusan.ac.kr; Tel.: +82-51-510-2395 Received: 12 December 2019; Accepted: 6 January 2020; Published: 10 January 2020 Abstract: Haematococcus pluvialis accumulates astaxanthin, which is a high-value antioxidant, during the red cyst stage of its lifecycle. The development of a rigid cell wall in the cysts hinders the recovery of astaxanthin. We investigated morphological changes and cell disruption of mature H. pluvialis cyst cells while using high-pressure homogenization for astaxanthin extraction. When treated with French-press-cell (pressure, 10,000–30,000 psi; passage, 1–3), the intact cyst cells were significantly broken or fully ruptured, releasing cytoplasmic components, thereby facilitating the separation of astaxanthin by ethyl acetate. Fluorescence microscopy observations using three di erent fluorescent dyes revealed that a greater degree of cell breakage caused greater external dispersion of astaxanthin, chlorophyll, lipids, proteins, and carbohydrates. The mechanical treatment resulted in a high cell disruption rate of up to 91% based on microscopic cell typing and Coulter methods. After the ethyl acetate extraction, the astaxanthin concentration significantly increased by 15.2 mg/L in proportion to the increase in cell disruption rate, which indicates that cell disruption is a critical factor for solvent-based astaxanthin recovery. Furthermore, this study recommends a synergistic combination of the fast instrumental particle-volume-distribution analysis and microscope-based morphologic phenotyping for the development of practical H. pluvialis biorefinery processes that co-produce various biological products, including lipids, proteins, carbohydrates, chlorophyll, and astaxanthin. Keywords: Haematococcus pluvialis; high-pressure homogenization; astaxanthin; cyst; cell disruption 1. Introduction Microalgal biomass is currently considered as a promising resource for valuable biochemicals and 0 0 biofuels [1,2]. Especially, astaxanthin (3,3 -dihydroxy- -carotene-4,4 -dione), which is a secondary ketocarotenoid compound, has been widely applied in feed, cosmetic, and pharmaceutical industries, owing to its excellent antioxidant, anti-inflammatory, and anti-cancer properties [3–5]. Although synthetic astaxanthin is extensively used as feed additives in aquaculture, natural astaxanthin is preferred over synthetic form for human consumption in view of its mixture of isomers and safety Appl. Sci. 2020, 10, 513; doi:10.3390/app10020513 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 513 2 of 10 concerns [6,7]. Several commercial-scale facilities currently produce natural astaxanthin from green microalga Haematococcus pluvialis having high astaxanthin content (~4%, w/w) [8,9]. H. pluvialis cell wall is dicult to destroy due to the considerable thickness and rigidity that are associated with its multiple layers formed during the transformation from green vegetative cells to astaxanthin-rich red cysts [10,11]. Apart from astaxanthin recovery, a biorefinery process that can co-produce other cellular components, such as lipids, proteins, and carbohydrates, can significantly reduce the cost of large-scale H. pluvialis-based astaxanthin production [12,13]. Accordingly, e orts to develop an environmental-friendly and cost-e ective cell disruption process from H. pluvialis is practically in progress. Various chemical and supercritical fluid extractions of astaxanthin from H. pluvialis biomass have been extensively studied [14,15]. However, the chemical treatment is potentially harmful with the excessive use of strong organic solvent (s) resulting in a lower stability and quality of astaxanthin [8]. The supercritical CO extraction process requires longer operation time and, generally, the use of co-solvent(s) and/or physicochemical pretreatment. Furthermore, this method is very expensive for large-scale biorefining applications [8,14]. High-pressure homogenization has been proposed as a relatively simple and importantly scalable cell disruption method for industrial-scale microalgal biorefining processes among various mechanical techniques, including bead milling, high-pressure homogenization, ultrafine grinding, autoclave, and sonication [8,12]. However, although there have been several studies on oleaginous Chlorella and Nannochloropsis species, especially for biodiesel production, application on harder aplanospore-forming species (e.g., H. pluvialis) is relatively limited [16]. In this study, the eciency of astaxanthin recovery according to cellular disruption level of H. pluvialis cyst cells was investigated by controlling high pressures (10,000–30,000 psi) and passages (1–3) of a French press cell as a model high-pressure homogenization method. During the mechanical cell disruption, the cell types were classified as intact, leaky, and ruptured based on optical microscopy. The size distribution of cellular particles and spatial distribution of intracellular biomolecules (e.g., astaxanthin, lipids, proteins, carbohydrates, and chlorophyll) were also characterized while using a Coulter counter and staining-based fluorescence microscopy techniques, respectively. Specific insights on their morphological changes and distributions will be beneficial for ecient scale-up and biorefinery applications. Finally, the technical feasibility of a fast instrumental Coulter counter was investigated as an alternative to the conventional time-consuming microscopic cell classification as an indicator of the algal cell disruption level [17]. 2. Materials and Methods 2.1. Materials Professor Sang Jun Sim of Korea University, Seoul, Korea provided the intact cysts of H. pluvialis NIES-144 that were used in this study. NIES-144 strain was originally obtained from the National Institute for Environmental Studies (NIES), Tsukuba, Japan, and cultivated in thin-film photobioreactors (working volume, ~63 L) under outdoor photoautotrophic conditions while using a CO -rich (~3.5%, v/v) power-plant flue gas (Korean District Heating Co., Pangyo, Gyeonggi, Korea). Comprehensive medium composition and culture conditions are available in the literature [18]. The H. pluvialis cysts were harvested by centrifugation (1100 g, 10 min., 4 C; Supra 22K, Hanil Science Inc., Gimpo, Korea), washed three times with purified water (Millipore Milli-Q system, Japan), and then freeze-dried for 48 h (FD5512, IlShin BioBase Co., Gyeonggi, Korea). The lyophilized H. pluvialis biomass was preserved in a sterile vacuum bag and stored at20 C in a dark environment until further use. All of the organic solvents and chemicals used in this study were of analytical grade and from Junsei (Tokyo, Japan) and Sigma Aldrich (St. Louis, MO, USA). Astaxanthin standard was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The fatty acid methyl ester (FAME) (Mix RM3, Mix RM5, GLC50, and GLC70) and sugar (D-glucose, D-arabinose, D-mannose, D-galactose, and D-xylose) standards were supplied from Sigma Aldrich (St. Louis, MO, USA). Appl. Sci. 2020, 10, 513 3 of 10 2.2. Mechanical Cell Disruption High-pressure homogenization method (French press cell, Thermo Electron Co., Waltham, MA, USA) was applied to algal biomass in three passes at three di erent pressures (10,000, 20,000, and 30,000 psi) to disrupt H. pluvialis cysts. The freeze-dried H. pluvialis biomass was dispersed in distilled water (Millipore Milli-Q system, Tokyo, Japan) at 1.0 g/L and vigorously mixed in a vortex (Vortex 3, IKA, Staufen, Germany) to ensure the homogeneity of the algal sample. The maximum internal capacity of the French press cell was 30 mL and the working volume in this study was fixed at 20 mL. In this process, the algal cells (1.0 g/L) are forced to flow through a very small orifice under high-pressure conditions, and, as a result, they could be disrupted by synergistic mechanical e ects, such as cavitation, turbulence, and shear stress [12]. 2.3. Microscopic Observation The cell disruption and distribution of internal biomolecules of H. pluvialis cyst cells were observed under a bright field and fluorescence microscope (Carl Zeiss imager A2, Oberkochen, Germany). In the bright field mode, after the high-pressure homogenization, H. pluvialis cysts containing astaxanthin (red color) could be largely classified into intact, leaky, and ruptured types while considering their morphological changes due to mechanical breakages. Cell staining was carried out in the order of proteins, carbohydrates, and lipids in order to simultaneously visualize the cell wall and various cytoplasmic components. To stain proteins, 100 L of sodium carbonate bu er (0.1 M) and 10 L of fluorescein isothiocyanate solution (FITC, 10 mg/mL) were added into the algal sample. Afterwards, the solution was stirred for 1 h at room temperature. Next, for carbohydrate staining, the cells were washed twice with phosphate bu er saline (pH = 7.2), and 100 L of Calcofluor white reagent (300 mg/L) was added thereto and then incubated for 30 min. Again, washing was performed twice with phosphate bu er saline (pH = 7.2), followed by the addition of 10 L of Nile red solution (20 mg/mL) for neutral lipid detection. Following this, the sample was incubated in a dark environment for 10 min. and then washed twice with phosphate bu er saline (pH = 7.2). In the fluorescent mode, the blue color of the carbohydrates was observed while using filter set 49, the green color of proteins and red color of chlorophyll auto-fluorescence was observed using filter set A9, and the yellow color of neutral lipids was observed using filter set 09. 2.4. Cell Disruption Eciency Estimation Instrumental particle-counting and microscopic cell typing methods evaluated cell disruption TM ecacy during the high-pressure homogenization. A Coulter counter (Multisizer 4, Beckman Coulter, Brea, CA, USA) with a 100 m aperture tube was used for determining the bio-volume, size, and number of particles in the range of 2–60 m. The sample volume was adjusted to 100 mL while using Isoton II electrolyte solution (Beckman Coulter, Brea, CA, USA) in the way that the particle concentration was kept below 10%. The cell disruption eciency (%) was estimated from the total bio-volume reduction degree of the homogenized sample versus the untreated control (100%). The total volume of particles was calculated by summing each volume of all the particles in the selected size range (especially 15–60 m). The cell disruption degree (i.e., the remaining intact type no/total cell no. after the mechanical treatment) was also estimated by manually sorting over 100 cells while using an improved Neubauer counting chamber (C-Chip, DHC-N01, iNCYTO, Chungnam, Korea) based on the optical microscope mentioned above. 2.5. Other Analytical Methods For astaxanthin extraction, 1 mL of French-press-treated H. pluvialis cells was vigorously mixed in a vortex mixer (Vortex 3, IKA, Germany) with 5 mL of ethyl acetate for 10 min. at room temperature. The mixture was separated by centrifugation (2000 g, 10 min.; Combi-514R, Hanil Science Inc., Gimpo, Korea) and an astaxanthin-containing organic fraction was collected and evaporated (N-EVAP Appl. Sci. 2020, 10, 513 4 of 10 evaporator; Organomation Associates Inc., Berlin, MA, USA). After saponification reaction with 0.025 N NaOH, free (de-esterified) astaxanthin content was analyzed while using a high-performance liquid chromatography (HPLC, Agilent 1260 series, Agilent, Santa Clara, CA, USA) that was equipped with a variable wavelength detector (VWD) and 250 4.6 mm YMC carotenoid column (Tokyo, Japan). The detailed extraction and HPLC conditions are detailed in the literature [6,19]. Lipid content of the freeze-dried H. pluvialis cysts was estimated by the FAME amount, according to a direct transesterification method, followed by gas chromatography (GC) [6,19]. Briefly, for lipid extraction, 2 mL of the chloroform/methanol (2:1, v/v) was added to the algal cells (~10 mg in a 12 mL Pyrex-glass tube). For the FAME analysis, subsequently, 1 mL of chloroform containing heptadecanoic acid (C17:0) as an internal standard (500 g/L), 1 mL of methanol, and 300 L of H SO (95%) were 2 4 added to the glass tube. The tube was incubated at 100 C for 10 min. (HS R200 heating-block reactor, Humas, Daejeon, Korea), cooled to room temperature, and then supplemented with 1 mL of distilled water. Finally, the FAME-containing organic phase was filtered by a PVDF (polyvinyl difluoride) syringe filter and then analyzed by GC that was equipped with a flame-ionization detector and a 0.32 mm (ID) 30 m HP-INNOWax capillary column (Agilent Technologies, Santa Clara, California, USA). The detailed GC conditions are available in [19]. Carbohydrate amount was estimated according to the NREL/TP-510-42618 protocol [20]. For cell hydrolysis, approximately 300 mg of the lyophilized H. pluvialis cells was mixed with 3 mL of H SO solution (72%) in a Pyrex-glass tube and then incubated for 60 min. at 30 C. Subsequently, 2 4 84 mL of purified water (Millipore Milli-Q system, Tokyo, Japan) was added and further incubated for 1 h at 121 C while using an autoclave (Napco 8000-DSE, Thermo Scientific Co., Waltham, MA, USA). The carbohydrate extract was cooled down to room temperature, centrifuged at 15,000 g for 5 min. (Combi-514R, Hanil Science Inc., Korea), and then PVDF syringe-filtered. Finally, the filtrate was analyzed using a HPLC equipped with a refractive-index (RI) detector (Agilent 1260, Agilent Co., Santa Clara, CA, USA) and Aminex HPX-87H column (300 7.8 mm, Bio-Rad, Hercules, CA, USA). As an eluent, 5 mM H SO solution was used at a flow rate of 0.5 mL/min. The temperatures of column 2 4 and RI detector were set at 60 C and 50 C, respectively. 3. Results and Discussion 3.1. Morphological Changes by Mechanical Disruption The e ect of French press cell treatment (10,000 psi, 1 pass) on mature H. pluvialis cysts were evaluated morpho-dynamically while using a combination of light and fluorescence microscopy (Figure 1). In the phase-contrast mode of optical microscope, the intact H. pluvialis cysts showed significant amounts of astaxanthin (bright red) and chlorophyll (green) inside the cells. The high-pressure homogenization could partially damage or completely rupture the cell walls of the cysts, resulting in significant releases of the cytoplasmic constituents into the surrounding medium. The H. pluvialis cysts could be largely categorized into intact, leaky, and ruptured types, depending on their degree of destruction (see Figure 2 for their distribution). Three di erent fluorescent probes of Calcofluor white, Nile red, and FITC were used under fluorescence microscopy, respectively, in order to simultaneously visualize the cell wall and cytoplasmic polysaccharides (blue), neutral lipids (yellow), and proteins (green) of H. pluvialis cyst cells. Figure 1 shows astaxanthin (magenta) and chlorophyll (red) auto-fluoresce without chemical staining. The total carbohydrate content of the H. pluvialis cysts that were incubated under the outdoor conditions while using a CO -rich (~3.5%, v/v) power-plant flue gas was estimated to be 12% (w/w). With the Calcofluor white dye, the polysaccharide fractions of the cell wall and the cytoplasmic contents were stained blue under the DAPI filter. The intact encysted cells showed a continuous blue color on their cell walls, because the H. pluvialis cell wall is mostly composed of mannan and cellulose polysaccharides [21,22]. However, significant dispersion of the blue-colored cell wall and cytoplasmic polysaccharides were observed after mechanical disruption. Appl. Sci. 2020, 10, 513 5 of 10 Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 10 Nile-red-stained neutral lipids are usually detected as cytoplasmic yellow-colored droplets in microscopy. The total lipid content of the H. pluvialis cyst cell was as high as 40% (w/w) when oleaginous microalgal species, such as Chlorella [23] and Nannochloropsis [24], under fluorescent estimated in terms of FAME amount by GC. This is similar to the typical lipid compositions of H. microscopy. The total lipid content of the H. pluvialis cyst cell was as high as 40% (w/w) when estimated pluvialis (32–37% (w/w)) harvested from the red cultivation stage [25]. However, it should be noted in terms of FAME amount by GC. This is similar to the typical lipid compositions of H. pluvialis (32–37% that, in H. pluvialis cysts, astaxanthin biomolecules co-exist inside the lipid body mostly in mono- and (w/w)) harvested from the red cultivation stage [25]. However, it should be noted that, in H. pluvialis di-ester forms with long-chain fatty acids [26,27]. Therefore, under the present fluorescence cysts, astaxanthin biomolecules co-exist inside the lipid body mostly in mono- and di-ester forms with microscopy condition, the former (magenta) and latter (yellow) cannot be clearly distinguished from long-chain fatty acids [26,27]. Therefore, under the present fluorescence microscopy condition, the the intact and leaky cell types. However, interestingly, after almost the complete rupture of the H. former (magenta) and latter (yellow) cannot be clearly distinguished from the intact and leaky cell pluvialis cyst cells, several yellow-colored lipid droplets appeared from the dispersed cytoplasm. The types. However, interestingly, after almost the complete rupture of the H. pluvialis cyst cells, several protein fractions from H. pluvialis cells fluoresced green after the FITC staining. As expected, the yellow-colored lipid droplets appeared from the dispersed cytoplasm. The protein fractions from H. intact cysts showed a relatively uniform green distribution in the cytosol, except for lipids, pluvialis cells fluoresced green after the FITC staining. As expected, the intact cysts showed a relatively astaxanthin, and chlorophyll. However, following the mechanical treatment, the intensity of gree n uniform green distribution in the cytosol, except for lipids, astaxanthin, and chlorophyll. However, color was significantly reduced. This might be mainly attributed to the significant loss of water- following the mechanical treatment, the intensity of green color was significantly reduced. This might soluble proteins in the medium. Overall, the degree of dispersion of the internal constituents, be mainly attributed to the significant loss of water-soluble proteins in the medium. Overall, the degree including astaxanthin, increased in proportion to the degree of cell rupture. This resulted in the of dispersion of the internal constituents, including astaxanthin, increased in proportion to the degree effective recovery of astaxanthin from the robust H. pluvialis cysts by subsequent solvent extraction of cell rupture. This resulted in the e ective recovery of astaxanthin from the robust H. pluvialis cysts (see next sections). by subsequent solvent extraction (see next sections). Figure 1. Microscopic images of H. pluvialis cysts after the high-pressure homogenization treatment (10,000 psi and one pass). Cell shapes were classified into three types (i.e., intact, leaky, and ruptured) Figure 1. Microscopic images of H. pluvialis cysts after the high-pressure homogenization treatment considering their morphological changes by the mechanical breakages. See colored arrows for each cell (10,000 psi and one pass). Cell shapes were classified into three types (i.e., intact, leaky, and ruptured) type. From left to right, optical phase-contrast images; fluorescent images under DAPI filter showing considering their morphological changes by the mechanical breakages. See colored arrows for each the cell wall and other polysaccharides (blue stained with Calcofluor white); fluorescent images under cell type. From left to right, optical phase-contrast images; fluorescent images under DAPI filter fluorescein isothiocyanate solution (FITC) long-pass filter showing the proteins (green stained with showing the cell wall and other polysaccharides (blue stained with Calcofluor white); fluorescent FITC), lipids (yellow stained with Nile red), chlorophyll (red by auto-fluorescence), and astaxanthin images under fluorescein isothiocyanate solution (FITC) long-pass filter showing the proteins (green (magenta by auto-fluorescence). stained with FITC), lipids (yellow stained with Nile red), chlorophyll (red by auto-fluorescence), and astaxanthin (magenta by auto-fluorescence). Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 10 3.2. Cell Type Distribution by Microscopic Counting Figure 2 shows the relative cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure intensity, 10,000–30,000 psi; pass, 1–3 times). The morphological cell types were calculated by sorting over 100 cells while using the improved Neubauer counting chamber under microscopic observation (also see Figure 1). After the homogenization in one pass at a pressure of 10,000 psi, the number of intact cyst cells accounted for ca. 45%, whereas the rest of the initial cysts became either leaky or ruptured completely (cell disruption, 54.8 ± 0.3%). Here, the cell disruption (%) was estimated by subtracting the % ratio of both the leaky and ruptured cells from the initial cells (100%). The cell disruption degree significantly increased to 80.2 ± 6.0% and 89 ± 0.8%, respectively, when the algal-suspension was homogenized once and twice more at the same pressure. On the contrary, at higher pressures of 20,000–30,000 psi, the cell disruption efficiency of H. pluvialis cysts did not improve when compared to that of the 10,000 psi treatment even if the passage number was increased from 1 to 3. The cell disruption efficacy of the high-pressure homogenization process can be influenced by the algal cell concentration, as well as the homogenizer specification (loading pressure and number of passage) [12]. Lowering the cell dosage might be considered in order to improve the cell disruption rate to more than 90%. However, it should be noted that this approach is not generally recommended in terms of large-scale biorefining of H. pluvialis biomass on an industrial scale [8]. Although the high-pressure homogenization has been recommended as a relatively efficient and scalable method among various physicochemical cell disruption techniques for large-scale algal biorefining [8,12], studies on H. pluvialis cyst cells are very limited. Praveenkumar et al. [19] reported that, when compared to ionic liquid-based extractions, the 30,000 psi-fixed high-pressure homogenization resulted in a high yield of 24 pg astaxanthin/cell from wet H. pluvialis cysts that were cultured under small-scale laboratory conditions. However, the cell disruption rate and key operating parameters (especially pressure intensity and number of passages) were not elucidated. Safi et al. [17] obtained the highest protein extraction yield (40%) from lyophilized H. pluvialis biomass while using a high-pressure cell disruptor that was fixed at 2700 bar (39,150 psi), as compared to manual grinding, alkaline treatment, and ultrasonication treatments. Moreover, they reported that, under the same condition, the maximal protein recovery efficiency showed a decreasing trend in accordance with the increasing order of cell wall rigidity among the tested algal strains: Arthrospira platensis < Porphyridium cruentum < Chlorella vulgaris < Nannochloropsis oculata < H. pluvialis. Detailed investigation into cell physiology, cell wall biology, and physical strength of H. pluvialis cells is required in order to develop an economical high-pressure homogenization process. In fact, this insight would create high potentialities for a synergistic or novel process that utilizes various existing biological–physicochemical Appl. Sci. 2020, 10, 513 6 of 10 treatments. Figure 2. Cell type distribution of H. pluvialis cysts after various high-pressure homogenization Figure 2. Cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure, 10,000–30,000 psi; passage, 1–3). The cell types were classified into intact, leaky, treatments (pressure, 10,000–30,000 psi; passage, 1–3). The cell types were classified into intact, leaky, and ruptured according to the morphological features that are based on the microscopic observation (Figure 1). 3.2. Cell Type Distribution by Microscopic Counting Figure 2 shows the relative cell type distribution of H. pluvialis cysts after various high-pressure homogenization treatments (pressure intensity, 10,000–30,000 psi; pass, 1–3 times). The morphological cell types were calculated by sorting over 100 cells while using the improved Neubauer counting chamber under microscopic observation (also see Figure 1). After the homogenization in one pass at a pressure of 10,000 psi, the number of intact cyst cells accounted for ca. 45%, whereas the rest of the initial cysts became either leaky or ruptured completely (cell disruption, 54.8 0.3%). Here, the cell disruption (%) was estimated by subtracting the % ratio of both the leaky and ruptured cells from the initial cells (100%). The cell disruption degree significantly increased to 80.2  6.0% and 89  0.8%, respectively, when the algal-suspension was homogenized once and twice more at the same pressure. On the contrary, at higher pressures of 20,000–30,000 psi, the cell disruption eciency of H. pluvialis cysts did not improve when compared to that of the 10,000 psi treatment even if the passage number was increased from 1 to 3. The cell disruption ecacy of the high-pressure homogenization process can be influenced by the algal cell concentration, as well as the homogenizer specification (loading pressure and number of passage) [12]. Lowering the cell dosage might be considered in order to improve the cell disruption rate to more than 90%. However, it should be noted that this approach is not generally recommended in terms of large-scale biorefining of H. pluvialis biomass on an industrial scale [8]. Although the high-pressure homogenization has been recommended as a relatively ecient and scalable method among various physicochemical cell disruption techniques for large-scale algal biorefining [8,12], studies on H. pluvialis cyst cells are very limited. Praveenkumar et al. [19] reported that, when compared to ionic liquid-based extractions, the 30,000 psi-fixed high-pressure homogenization resulted in a high yield of 24 pg astaxanthin/cell from wet H. pluvialis cysts that were cultured under small-scale laboratory conditions. However, the cell disruption rate and key operating parameters (especially pressure intensity and number of passages) were not elucidated. Safi et al. [17] obtained the highest protein extraction yield (40%) from lyophilized H. pluvialis biomass while using a high-pressure cell disruptor that was fixed at 2700 bar (39,150 psi), as compared to manual grinding, alkaline treatment, and ultrasonication treatments. Moreover, they reported that, under the same condition, the maximal protein recovery eciency showed a decreasing trend in accordance with the increasing order of cell wall rigidity among the tested algal strains: Arthrospira platensis < Porphyridium cruentum < Chlorella vulgaris < Nannochloropsis oculata < H. pluvialis. Detailed investigation into cell physiology, cell wall biology, and physical strength of H. pluvialis cells is required in order to develop an Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 10 Appl. Sci. 2020, 10, 513 7 of 10 and ruptured according to the morphological features that are based on the microscopic observation (Figure 1). economical high-pressure homogenization process. In fact, this insight would create high potentialities for a synergistic or novel process that utilizes various existing biological–physicochemical treatments. 3.3. Bio-Particle Distribution by Coulter Counter and Astaxanthin Recovery 3.3. Bio-Particle Distribution by Coulter Counter and Astaxanthin Recovery Figure 3 shows the bio-volume distribution and total bio-volume of H. pluvialis bio-particles before and after French press cell treatment with different pressures (10,000–30,000 psi) and then Figure 3 shows the bio-volume distribution and total bio-volume of H. pluvialis bio-particles before passes (1–3). Here, “bio-particles” collectively refer to the ruptured cells, cell debris, and internal and after French press cell treatment with di erent pressures (10,000–30,000 psi) and then passes (1–3). biomolecules that have leaked outwards, as well as the initial intact cells. In the untreated control, Here, “bio-particles” collectively refer to the ruptured cells, cell debris, and internal biomolecules that most of the H. pluvialis cysts were of 15–60 µ m cell size and they exhibited a relatively large particle have leaked outwards, as well as the initial intact cells. In the untreated control, most of the H. pluvialis 7 3 volume distribution (~4.1 × 10 µm /mL) (Figure 3a). However, when homogenized at a pressure of cysts were of 15–60 m cell size and they exhibited a relatively large particle volume distribution 10,000 psi, the particle volumes of bio-particles in the same range significantly decreased in 7 3 (~4.1 10 m /mL) (Figure 3a). However, when homogenized at a pressure of 10,000 psi, the particle 7 3 7 3 proportion to the increase in the number of passage: ~1.7 × 10 µm /mL for one pass, ~1.3 × 10 µm /mL volumes of bio-particles in the same range significantly decreased in proportion to the increase in 7 3 for two passes, and ~0.5 × 10 µm /mL for three passes. On the contrary, the volumes of the bio- 7 3 7 3 the number of passage: ~1.7  10 m /mL for one pass, ~1.3  10 m /mL for two passes, and particles smaller than ~15 µ m increased slightly when compared to the untreated control. These 7 3 ~0.5  10 m /mL for three passes. On the contrary, the volumes of the bio-particles smaller than changes were induced by the mechanical destruction of the initial cyst cells (see Figure 1 for ~15 m increased slightly when compared to the untreated control. These changes were induced by morphological changes). the mechanical destruction of the initial cyst cells (see Figure 1 for morphological changes). Figure 3. Changes in volume distribution (a) and total volume (b) of H. pluvialis bio-particles after Figure 3. Changes in volume distribution (a) and total volume (b) of H. pluvialis bio-particles after mechanical disruptions with di erent pressures and passes. The bio-particles refer to all internal mechanical disruptions with different pressures and passes. The bio-particles refer to all internal components as well as intact, leaky, and ruptured cyst cells. Representative particle-size-volume components as well as intact, leaky, and ruptured cyst cells. Representative particle-size-volume distributions after the 10,000 psi-homogenizations at three di erent passes were presented based on distributions after the 10,000 psi-homogenizations at three different passes were presented based on the Coulter counter analysis. Total volumes were calculated by summing each volume of all particles the Coulter counter analysis. Total volumes were calculated by summing each volume of all particles sized between 15 and 60 m. sized between 15 and 60 µ m. Figure 3b plotted the changes in the total bio-volumes of the bio-particles after the homogenization Figure 3b plotted the changes in the total bio-volumes of the bio-particles after the treatments. The total bio-volume of the homogenized bio-particles was calculated by summing homogenization treatments. The total bio-volume of the homogenized bio-particles was calculated the volume of each bio-particle between 15 to 60 m while considering their characteristic volume by summing the volume of each bio-particle between 15 to 60 µ m while considering their 9 3 reductions. The total bio-volume of the untreated H. pluvialis cysts (1.9  10 m /mL) decreased characteristic volume reductions. The total bio-volume of the untreated H. pluvialis cysts (1.9 × 10 almost linearly with an increasing number of passes (1–3), irrespective of the tested pressure intensity m /mL) decreased almost linearly with an increasing number of passes (1–3), irrespective of the tested (10,000–30,000 psi). However, no significant positive e ect of the high pressures (20,000–30,000 psi) was pressure intensity (10,000–30,000 psi). However, no significant positive effect of the high pressures observed as compared to the case of 10,000 psi, which is similar to that of the microscopic cell typing (20,000–30,000 psi) was observed as compared to the case of 10,000 psi, which is similar to that of the (Figure 2). The cell disruption eciency could be simply derived from the degree of total volume microscopic cell typing (Figure 2). The cell disruption efficiency could be simply derived from the reduction of the homogenized samples (versus the untreated control, 100%), and its maximal value degree of total volume reduction of the homogenized samples (versus the untreated control, 100%), was estimated to be 91.8 2.2%. This value was also almost equal to the highest value (89 0.8%) that and its maximal value was estimated to be 91.8 ± 2.2%. This value was also almost equal to the highest was obtained from the microscopic cell counting in Figure 2. value (89 ± 0.8%) that was obtained from the microscopic cell counting in Figure 2. When comparing the cell disruption eciencies that were calculated from Coulter counting and When comparing the cell disruption efficiencies that were calculated from Coulter counting and microscopic cell typing methods, a statistically good correlation (r = 0.95) was obtained (Figure 4a). microscopic cell typing methods, a statistically good correlation (r = 0.95) was obtained (Figure 4a). Furthermore, by the subsequent ethyl acetate extraction, astaxanthin was recovered from the H. pluvialis Furthermore, by the subsequent ethyl acetate extraction, astaxanthin was recovered from the H. biomass treated under various high-pressure homogenization conditions (see Figures 2 and 3). As pluvialis biomass treated under various high-pressure homogenization conditions (see Figures 2 and Appl. Sci. 2020, 10, 513 8 of 10 Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 10 3). As expected, the astaxanthin concentration increased almost linearly from 5.3 to 15.2 mg/L in expected, the astaxanthin concentration increased almost linearly from 5.3 to 15.2 mg/L in proportion proportion to the increase in cell disruption level (Figure 4b), which indicated that cell disruption is to the increase in cell disruption level (Figure 4b), which indicated that cell disruption is a critical a critical factor for solvent-based astaxanthin recovery. The astaxanthin content of the H. pluvialis factor for solvent-based astaxanthin recovery. The astaxanthin content of the H. pluvialis biomass was biomass was estimated to be 1.1% (w/w). Astaxanthin from the freeze-dried H. pluvialis cyst cells estimated to be 1.1% (w/w). Astaxanthin from the freeze-dried H. pluvialis cyst cells could also be could also be extracted while using solely ethyl acetate, although its concentration was as low as 5.3 extracted while using solely ethyl acetate, although its concentration was as low as 5.3 mg/L. The mg/L. The statistical r values for relationships between the astaxanthin recovery and cell disruption statistical r values for relationships between the astaxanthin recovery and cell disruption degree degree were estimated to be 0.83 and 0.86 for the microscopic and Coulter counter methods, were estimated to be 0.83 and 0.86 for the microscopic and Coulter counter methods, respectively. respectively. This result suggests that the Coulter counter method can be used effectively for the rapid This result suggests that the Coulter counter method can be used e ectively for the rapid assessment assessment of H. pluvialis cell disruption for astaxanthin recovery. Microscopy phenotyping generally of H. pluvialis cell disruption for astaxanthin recovery. Microscopy phenotyping generally requires requires time-consuming and laborious experimentation by skilled personnel. However, it should be time-consuming and laborious experimentation by skilled personnel. However, it should be noted that noted that this approach is very useful in understanding the actual morphological changes of the this approach is very useful in understanding the actual morphological changes of the rigid H. pluvialis rigid H. pluvialis cysts and the spatial distribution of the target biomolecules during mechanical cysts and the spatial distribution of the target biomolecules during mechanical treatment (Figure 1). treatment (Figure 1). Therefore, a synergistic combination of these methods is recommended for the Therefore, a synergistic combination of these methods is recommended for the development of practical development of practical H. pluvialis biorefinery processes that co-produce various bio-products, H. pluvialis biorefinery processes that co-produce various bio-products, including lipids, proteins, including lipids, proteins, carbohydrates, chlorophyll, and astaxanthin. Several analytical techniques carbohydrates, chlorophyll, and astaxanthin. Several analytical techniques have been reported in have been reported in order to evaluate the disintegration efficiency of algal biomass by mechanical order to evaluate the disintegration eciency of algal biomass by mechanical treatments: particle-size treatments: particle-size analyzer (homogenization for Chlamydomonas reinhardtii and analyzer (homogenization for Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata [28]; bead Pseudokirchneriella subcapitata [28]; bead beating for C. reinhardtii [29]), dynamic light scattering beating for C. reinhardtii [29]), dynamic light scattering (ultrasonication for Parachlorella kessleri [30]), (ultrasonication for Parachlorella kessleri [30]), and flow cytometer (bead milling for Chlorella species and flow cytometer (bead milling for Chlorella species [31,32]). However, after cell disruption, the [31,32]). However, after cell disruption, the morphological characteristics of the algal cell and/or the morphological characteristics of the algal cell and/or the internal target substance(s) have not been internal target substance(s) have not been studied in detail. studied in detail. Figure 4. (a) Cell disruption eciency relationship between microscopic cell typing and instrumental Figure 4. (a) Cell disruption efficiency relationship between microscopic cell typing and instrumental particle-size-distribution measurements (r = 0.95); (b) astaxanthin recovery, depending on cell particle-size-distribution measurements (r = 0.95); (b) astaxanthin recovery, depending on cell 2 2 disruption eciencies (microscope, r = 0.83; Coulter counter, r = 0.86). 2 2 disruption efficiencies (microscope, r = 0.83; Coulter counter, r = 0.86). 4. Conclusions 4. Conclusions High-pressure homogenization might be used for highly ecient cell disruption and astaxanthin High-pressure homogenization might be used for highly efficient cell disruption and recovery from mature H. pluvialis cyst cells, followed by ethyl acetate extraction. French press cell astaxanthin recovery from mature H. pluvialis cyst cells, followed by ethyl acetate extraction. French treatment e ectively destroys the H. pluvialis cyst cells and results in a high disruption rate of up to 91%. press cell treatment effectively destroys the H. pluvialis cyst cells and results in a high disruption rate The homogenized cells can be classified into intact, leaky, and completely ruptured, according to their of up to 91%. The homogenized cells can be classified into intact, leaky, and completely ruptured , morphological changes under an optical microscope. The degree of external dispersion of astaxanthin, according to their morphological changes under an optical microscope. The degree of external lipids, proteins, and carbohydrates was almost proportional to the degree of cell rupture when analyzed dispersion of astaxanthin, lipids, proteins, and carbohydrates was almost proportional to the degree by fluorescence microscopy with specific fluorescent probes. Pressure intensity (10,000–30,000 psi) did of cell rupture when analyzed by fluorescence microscopy with specific fluorescent probes. Pressure not a ect the cell disruption eciency, but an increasing the number of passages (1–3 times) could intensity (10,000–30,000 psi) did not affect the cell disruption efficiency, but an increasing the number significantly improve the cell disruption eciency. After the subsequent ethyl acetate extractions, of passages (1–3 times) could significantly improve the cell disruption efficiency. After the the astaxanthin concentration increased linearly from 5.3 to 15.2 mg/L with increased cell disruption subsequent ethyl acetate extractions, the astaxanthin concentration increased linearly from 5.3 to 15.2 mg/L with increased cell disruption efficacy owing to homogenization optimization. The maximum astaxanthin recovery was estimated to be 1.1% (weight of dry cells). Appl. Sci. 2020, 10, 513 9 of 10 ecacy owing to homogenization optimization. The maximum astaxanthin recovery was estimated to be 1.1% (weight of dry cells). Author Contributions: Conceptualization, R.P. and K.L.; methodology, R.P., J.L., D.V., M.E.H. and S.J.S.; Writing—Original draft preparation, R.P.; Writing—Review and editing, R.P., Y.-K.O., Y.-E.K. and S.Y.L.; supervision, Y.-K.O.; funding acquisition, Y.-K.O. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Research Foundation of Korea (grant number: NRF-2019R1A2C1003463) funded by the Ministry of Science and ICT and the Research/Development Program of the Korea Institute of Energy Research (grant number: KIER-B9-2442-04). Sim, S.J., also would like to acknowledge the support of the Korea CCS R & D Center (Korea CCS 2020 Project) funded by the Ministry of Science and ICT in 2017 (grant number: KCRC-2014M1A8A1049278). Conflicts of Interest: The authors declare no conflict of interest. References 1. Bauer, A.; Minceva, M. Direct extraction of astaxanthin from the microalgae: Haematococcus pluvialis using liquid-liquid chromatography. RSC Adv. 2019, 9, 22779–22789. [CrossRef] 2. Matter, I.A.; Hoang Bui, V.K.; Jung, M.; Seo, J.Y.; Kim, Y.E.; Lee, Y.C.; Oh, Y.K. Flocculation harvesting techniques for microalgae: A review. Appl. Sci. 2019, 9, 3069. [CrossRef] 3. Focsan, A.L.; Polyakov, N.E.; Kispert, L.D. Photo protection of Haematococcus pluvialis algae by astaxanthin: Unique properties of astaxanthin deduced by EPR, optical and electrochemical studies. Antioxidants 2017, 6, 80. [CrossRef] [PubMed] 4. Guerin, M.; Huntley, M.E.; Olaizola, M. Haematococcus astaxanthin: Applications for human health and nutrition. Trends Biotechnol. 2003, 21, 210–216. [CrossRef] 5. Pérez-López, P.; González-García, S.; Je ryes, C.; Agathos, S.N.; McHugh, E.; Walsh, D.; Murray, P.; Moane, S.; Feijoo, G.; Moreira, M.T. Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: From lab to pilot scale. J. Clean. Prod. 2014, 64, 332–344. [CrossRef] 6. Choi, S.A.; Oh, Y.K.; Lee, J.; Sim, S.J.; Hong, M.E.; Park, J.Y.; Kim, M.S.; Kim, S.W.; Lee, J.S. High-eciency cell disruption and astaxanthin recovery from Haematococcus pluvialis cyst cells using room-temperature imidazolium-based ionic liquid/water mixtures. Bioresour. Technol. 2019, 274, 120–126. [CrossRef] [PubMed] 7. Samorì, C.; Pezzolesi, L.; Galletti, P.; Semeraro, M.; Tagliavini, E. Extraction and milking of astaxanthin from: Haematococcus pluvialis cultures. Green Chem. 2019, 21, 3621–3628. [CrossRef] 8. Kim, D.Y.; Vijayan, D.; Praveenkumar, R.; Han, J.I.; Lee, K.; Park, J.Y.; 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. [CrossRef] 9. 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. [CrossRef] 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. [CrossRef] 11. Machado, F.R.S.; Trevisol, T.C.; Boschetto, D.L.; Burkert, J.F.M.; Ferreira, S.R.S.; Oliveira, J.V.; Burkert, C.A.V. Technological process for cell disruption, extraction and encapsulation of astaxanthin from Haematococcus pluvialis. J. Biotechnol. 2016, 218, 108–114. [CrossRef] [PubMed] 12. Lee, S.Y.; Cho, J.M.; Chang, Y.K.; Oh, Y.K. Cell disruption and lipid extraction for microalgal biorefineries: A review. Bioresour. Technol. 2017, 244, 1317–1328. [CrossRef] [PubMed] 13. Khoo, K.S.; Lee, S.Y.; Ooi, C.W.; Fu, X.; Miao, X.; Ling, T.C.; Show, P.L. Recent advances in biorefinery of astaxanthin from Haematococcus pluvialis. Bioresour. Technol. 2019, 121606. [CrossRef] [PubMed] 14. Reyes, F.A.; Mendiola, J.A.; Ibañez, E.; Del Valle, J.M. Astaxanthin extraction from Haematococcus pluvialis using CO -expanded ethanol. J. Supercrit. Fluids 2014, 92, 75–83. [CrossRef] 15. Sarada, R.; Vidhyavathi, R.; Usha, D.; Ravishankar, G.A. An ecient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J. Agric. Food Chem. 2006, 54, 7585–7588. [CrossRef] 16. Park, J.Y.; Oh, Y.K.; Choi, S.A.; Kim, M.C. Recovery of astaxanthin-containing oil from Haematococcus pluvialis by nano-dispersion and oil partitioning. Appl. Biochem. Biotechnol. 2020, 1–15. [CrossRef] Appl. Sci. 2020, 10, 513 10 of 10 17. Safi, C.; Ursu, A.V.; Laroche, C.; Zebib, B.; Merah, O.; Pontalier, P.Y.; Vaca-Garcia, C. Aqueous extraction of proteins from microalgae: E ect of di erent cell disruption methods. Algal Res. 2014, 3, 61–65. [CrossRef] 18. Choi, Y.Y.; Hong, M.E.; Jin, E.S.; Woo, H.M.; Sim, S.J. Improvement in modular scalability of polymeric thin-film photobioreactor for autotrophic culturing of Haematococcus pluvialis using industrial flue gas. Bioresour. Technol. 2018, 249, 519–526. [CrossRef] 19. Praveenkumar, R.; Lee, K.; Lee, J.; Oh, Y.K. Breaking dormancy: An energy-ecient means of recovering astaxanthin from microalgae. Green Chem. 2015, 17, 1226–1234. [CrossRef] 20. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. NREL/TP-510-42618 analytical procedure—Determination of structural carbohydrates and lignin in biomass. Lab. Anal. Proced. 2008, 1617, 1–16. 21. Hagen, C.; Siegmund, S.; Braune, W.; Botanik, A.; Jena, F.; Planetarium, A. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur. J. Phycol. 2002, 37, 217–226. [CrossRef] 22. Damiani, M.C.; Leonardi, P.I.; Pieroni, O.I.; Cáceres, E.J. Ultrastructure of the cyst wall of Haematococcus pluvialis (Chlorophyceae): Wall development and behaviour during cyst germination. Phycologia 2006, 45, 616–623. [CrossRef] 23. Lee, Y.C.; Lee, H.U.; Lee, K.; Kim, B.; Lee, S.Y.; Choi, M.H.; Farooq, W.; Choi, J.S.; Park, J.Y.; Lee, J.; et al. Aminoclay-conjugated TiO synthesis for simultaneous harvesting and wet-disruption of oleaginous Chlorella sp. Chem. Eng. J. 2014, 245, 143–149. [CrossRef] 24. Wei, L.; Huang, X. Long-duration e ect of multi-factor stresses on the cellular biochemistry, oil-yielding performance and morphology of Nannochloropsis oculata. PLoS ONE 2017, 12, e0174646. [CrossRef] [PubMed] 25. Shah, M.M.R.; Liang, Y.; Cheng, J.J.; Daroch, M. Astaxanthin-producing green microalga Haematococcus pluvialis: From single cell to high value commercial products. Front. Plant Sci. 2016, 7, 531. [CrossRef] 26. Ding, W.; Li, Q.; Han, B.; Zhao, Y.; Geng, S.; Ning, D. Comparative physiological and metabolomic analyses of the hyper-accumulation of astaxanthin and lipids in Haematococcus pluvialis upon treatment with butylated hydroxyanisole. Bioresour. Technol. 2019, 292, 122002. [CrossRef] [PubMed] 27. Peled, E.; Leu, S.; Zarka, A.; Weiss, M.; Pick, U.; Khozin-Goldberg, I.; Boussiba, S. Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (chlorophyceae). Lipids 2011, 46, 851–861. [CrossRef] 28. Lavoie, M.; Bernier, J.; Fortin, C.; Campbell, P.G.C. Cell homogenization and subcellular fractionation in two phytoplanktonic algae: Implications for the assessment of metal subcellular distributions. Limnol. Oceanogr. Methods 2009, 7, 277–286. [CrossRef] 29. Lam, G.P.; Van Der Kolk, J.A.; Chordia, A.; Vermuë, M.H.; Olivieri, G.; Eppink, M.H.M.; Wij els, R.H. Mild and selective protein release of cell wall deficient microalgae with pulsed electric field. ACS Sustain. Chem. Eng. 2017, 5, 6046–6053. [CrossRef] 30. Piasecka, A.; Ciesla, ´ J.; Koczanska, ´ M.; Krzeminska, ´ I. E ectiveness of Parachlorella kessleri cell disruption evaluated with the use of laser light scattering methods. J. Appl. Phycol. 2019, 31, 97–107. [CrossRef] 31. Postma, P.R.; Miron, T.L.; Olivieri, G.; Barbosa, M.J.; Wij els, R.H.; Eppink, M.H.M. Mild disintegration of the green microalgae Chlorella vulgaris using bead milling. Bioresour. Technol. 2015, 184, 297–304. [CrossRef] [PubMed] 32. Günerken, E.; D’Hondt, E.; Eppink, M.; Elst, K.; Wij els, R. Flow cytometry to estimate the cell disruption yield and biomass release of Chlorella sp. during bead milling. Algal Res. 2017, 25, 25–31. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Journal

Applied SciencesMultidisciplinary Digital Publishing Institute

Published: Jan 10, 2020

There are no references for this article.