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Article

Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts

by
Catherine Christabel
1,
Bolam Kim
2,3,
Aditya Lakshmi Narasimhan
1,
Laxmi Priya Sathiyavahisan
1,
Dea Prianka Ayu Ilhamsyah
1,4,
Eui-Jin Kim
5 and
You-Kwan Oh
1,2,*
1
School of Chemical Engineering, Pusan National University, Busan 46241, Republic of Korea
2
Institute of Environment & Energy, Pusan National University, Busan 46241, Republic of Korea
3
Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea
4
School of Chemical and Biomolecular Engineering, University of Sydney, Sydney 2006, Australia
5
Bioresources Research Division, Nakdonggang National Institute of Biological Resources, Sangju 37242, Republic of Korea
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(22), 10434; https://doi.org/10.3390/app142210434
Submission received: 29 September 2024 / Revised: 4 November 2024 / Accepted: 12 November 2024 / Published: 13 November 2024
(This article belongs to the Section Applied Biosciences and Bioengineering)
Browse Figures
Versions Notes

Abstract

:
The slow growth and complex life cycle of Haematococcus lacustris pose significant challenges for cost-effective astaxanthin production. This study explores the use of microfluidic collision treatment to stimulate the germination of dormant seed cysts, thereby improving photosynthetic cell growth and astaxanthin productivity in H. lacustris cultivated in well plate and flask cultures. The flow rate (1.0–3.0 mL/min) and the number of T-junction loops (3–30) were optimized in the microfluidic device. Under optimal conditions (a flow rate of 2.0 mL/min with 10 loops), the total cell number density in well plate cultures increased by 44.5% compared to untreated controls, reaching 28.9 ± 2.0 × 104 cells/mL after 72 h. In flask cultures, treated cysts showed a 21% increase in astaxanthin productivity after 30 d, reaching 0.95 mg/L/d, due to higher biomass concentrations, while the astaxanthin content per cell remained constant. However, excessive physical collision stress at higher flow rates and loop numbers resulted in reduced cell viability and cell damage. These findings suggest that carefully controlled cyst mechanostimulation can be an effective and environmentally friendly strategy for Haematococcus biorefining, enabling the production of multiple bioactive products.

1. Introduction

Microalgae perform photosynthesis using only carbon dioxide (CO2) and light as carbon and energy sources, respectively [1,2,3]. Unlike traditional crops, microalgae do not require arable land and can utilize wastewater and CO2-rich exhaust gases, making them an environmentally sustainable and versatile resource for various industries, including functional foods, pharmaceuticals, animal feed, biofuels, biochemicals, and CO2 mitigation [4,5].
Astaxanthin is one of the valuable compounds produced by microalgae. Astaxanthin is a secondary ketocarotenoid pigment that is particularly notable for its powerful antioxidant properties, which surpass those of β-carotene and vitamin C [6,7]. Astaxanthin has wide applications in the aquaculture, cosmetics, pharmaceutical, nutraceutical, and food sectors [8,9]. Currently, most astaxanthin is produced through chemical synthesis, but concerns about the safety of synthetic astaxanthin for human consumption have driven interest in natural sources [10,11]. The green microalga Haematococcus lacustris (formally known as H. pluvialis; [12]) is extensively cultivated for its high astaxanthin content, which can reach up to 5% of its dry weight, to meet the growing global demand for natural astaxanthin [7,13].
The life cycle of H. lacustris is complex, encompassing three distinct stages: cyst germination, vegetative cell growth, and encystment, the latter being associated with astaxanthin production [14,15]. Astaxanthin biosynthesis is triggered by reactive oxygen species (ROS) signaling, serving as a defense mechanism against unfavorable conditions [7,16]. However, the photosynthetic cultivation of H. lacustris is time-consuming, often requiring more than two months [17]. Strategies explored to enhance astaxanthin productivity include inducing cyst germination with higher nitrate concentrations [18,19] or blue light-emitting diode (LED) light [20]. Additionally, conventional photosynthetic parameters, such as CO2 supply, light intensity, pH, temperature, and media composition, have been extensively optimized [6,21,22].
A range of abiotic stresses, including intense light [23], nutrient deficiency [24], high salinity [25], elevated temperatures [26], the addition of aminoclay nanoparticles [27], phytohormone supplementation [28], hydrostatic pressure [29], electrical treatment [30], and ultrasonication [31], have also been employed to stimulate encystment and enhance astaxanthin production in H. lacustris. However, many of these methods are costly, energy-intensive, or involve chemicals unsuitable for human and animal consumption. Consequently, developing effective and environmentally friendly strategies to enhance microalgal astaxanthin production remains a significant challenge for large-scale applications [16,32].
Mechanostimulation, which involves applying mechanical or physical stimuli that include pressure, tension, compression, and shear stress, has emerged as a promising approach [33,34]. These mechanical stresses on the cell membrane can influence various biological processes, including cytoskeleton dynamics, protein conformation, cell division, cell cycle progression, gene expression, and protein synthesis [35,36]. Notably, mechanostimulation offers environmental benefits for microalgal applications, operating under ambient conditions without the need for chemical additives. For example, Min et al. [35] reported enhanced cell division in the green microalga Chlamydomonas reinhardtii under microfluidic compression, which led to increased production of biflagellate daughter cells after 96 h. However, the effects of mechanical stress can vary significantly depending on the organism, the intensity of the stress, and the specific methodology employed [37].
For instance, Han et al. [38] and Yao et al. [39] developed a microfabricated culture platform that physically compressed vegetative biflagellate cells of H. lacustris. The compression increased ROS levels and resulted in a ninefold increase in astaxanthin accumulation after 7 d of cultivation. However, this approach also inhibited photosynthetic cell growth due to the prolonged and excessive stress applied.
Building on these insights and considering the unique cell cycle of H. lacustris, this study aimed to investigate the feasibility of using short and mild mechanical collisions to stimulate the germination of dormant cysts, which can then serve as inoculum for photosynthetic astaxanthin production. We hypothesize that faster cyst germination will promote accelerated photosynthetic growth, thereby enhancing astaxanthin productivity. To test this, we employed a microfluidic chip design, inspired by Deng et al. [40]. The chip features loops and sharp T-junctions to regulate the intensity of mechanical stimulation across varying flow rates (1.0 to 3.0 mL/min) and loop numbers (3 to 30). The impact of these collisions on the viability and ROS levels of H. lacustris cysts was assessed. Germination efficiency, cell growth, and astaxanthin content and productivity were evaluated in photosynthetic well plate and flask cultures over 3 d and 30 d, respectively.

2. Materials and Methods

2.1. Microalga and Photosynthetic Cultivation

H. lacustris strain NIES-144 originated from the National Institute for Environmental Studies (NIES), Tsukuba, Japan. Photoautotrophic cultivation was conducted in NIES-C medium (pH 7.5) using 250 mL Erlenmeyer flasks with a working volume of 150 mL that were sealed with a porous stopper. The flasks were incubated in a model ISF-7100RF shaking incubator (JEIO TECH, Daejeon, Republic of Korea) at 150 rpm and 25 °C. Continuous illumination was provided by three white LED lamps at a light intensity of 80 ± 5 μmol photons/m2/s. The NIES-C medium was prepared with the following composition: Ca(NO3)2 0.15 g/L, KNO3 0.10 g/L, β-glycerophosphoric acid disodium salt pentahydrate 0.05 g/L, MgSO4·7H2O 0.04 g/L, tris-aminomethane 0.50 g/L, thiamine 0.01 mg/L, biotin 0.10 μg/L, vitamin B12 0.10 μg/L, and PIV metal solution 3.0 mL/L. The PIV metal solution contained the following: Na2EDTA 1.0 g/L, FeCl3·6H2O 0.196 g/L, MnCl2·4H2O 36.0 mg/L, ZnSO4·7H2O 22.0 mg/L, CoCl2·6H2O 4.0 mg/L, and Na2MoO4·2H2O 2.5 mg/L [32].

2.2. Microfluidic Collision and Experimentation

The process of mechanically colliding H. lacustris cysts using a microfluidic system is illustrated in Figure 1. The microfluidic chip was fabricated via conventional photolithography based on a previously established protocol [41,42]. Briefly, the chip mold was designed in AutoCAD® (Autodesk, San Francisco, CA, USA) with straight-line microchannels for cell lining and T-junction loop structures for cell collision (Deng et al. [40]). The design was printed on photoresist negative film and used in photolithography. Silicon wafers were coated with SU-8 50 negative epoxy photoresist and pre-baked, exposed to ultraviolet light, post-baked, developed, washed, and cured to prepare the mold.
The microfluidic device was fabricated by mixing polydimethylsiloxane (PDMS) with silicone elastomer hardener (10:1 ratio; Dow Chemical Co., Midland, MI, USA), pouring the mixture into the mold, and baking it at 70 °C for 2 h. After curing, the PDMS slab was peeled off, and inlets and outlets were punched using a sterile punch (Kai Medical, Seki, Japan). The slab was then irreversibly bonded to a glass coverslip via plasma treatment using a model PDC-32G device (Harrick Plasma, Ithaca, NY, USA) and further baked at 70 °C for 8 h to strengthen the bond.
Mature red cysts of H. lacustris (approximately 0.2 g/L), harvested from 33-d-old seed cultures, were washed twice with fresh NIES-C medium before injection into the microfluidic device. The injection was performed using a Legato 101 syringe pump (KD Scientific, Holliston, MA, USA) equipped with a 6 mL Norm-ject® syringe (Henke-Sass, Wolf GmbH, Tuttlingen, Germany) and a sterile 23G needle (Korea Vaccine Co., Seoul, Republic of Korea), connected to the device inlet via Tygon® tubing (0.02-inch inner diameter; Saint Gobain PPL, Taipei, Taiwan). The outlet was similarly connected for cell collection.
Mechanical collision intensity was adjusted by varying the flow rate (1.0 to 3.0 mL/min) and the number of T-junction loop passages (3 to 30). The treated cysts were cultivated photosynthetically in either 24-well plates (2 mL working volume) for 3 d or 250 mL Erlenmeyer flasks (120 mL working volume) for 30 d under the same conditions as the seed flask cultures. Controls involved passing cysts through Tygon® (Saint-Gobain Performance Plastic’s, Cleveland, ON, USA) tubing without microfluidic collision.

2.3. Cell Morphology and Viability Analyses

Time-lapse imaging of microalgal cells was performed by bright-field microscopy using an Axio Vert.A1 microscope (Carl Zeiss, Jena, Germany) equipped with an Axio 305 digital camera (Carl Zeiss) and Zeiss Zen Blue imaging software (Version 3.8; Carl Zeiss). Images were captured every 15 min from 12 to 72 h, and time-lapse video was recorded (see Supplementary Video S1). Cell number density was determined using a Neubauer hemocytometer (Mariendelf, Lauda-Königshofen, Germany). Cell viability (%) was accessed microscopically using trypan blue dye, which penetrates the damaged cytoplasmic membranes of H. lacustris based on a previously described protocol [43].

2.4. Quantification of ROS

Cellular ROS levels were estimated using 2′7′-dichlorofluorescein diacetate (DCFDA) as a cell-membrane-permeable fluorogenic probe, as previously described [43]. After microfluidic treatment, H. lacustris cell density was adjusted to approximately 1 × 106 cells/mL. The suspension was centrifuged at 10,000× g for 5 min, washed with 0.1 M phosphate-buffered saline (PBS; pH 7.0), and incubated with DCFDA dye in a shaking incubator at 37 °C for 1 h in the dark. Cells were then washed and analyzed by fluorescence spectrophotometry using a Wallac Victor2 1420 Multilabel Counter with excitation and emission wavelengths of 485 and 535 nm, respectively (PerkinElmer Life Sciences, Waltham, MA, USA).

2.5. Quantification of Astaxanthin

Cellular astaxanthin content was measured using bead-beating-assisted solvent extraction and high-performance liquid chromatography (HPLC), following the protocols of Mahadi et al. [29] and Lakshmi Narasimhan et al. [32]. After 30 d of photosynthetic cultivation, H. lacustris cells were harvested by centrifugation at 1952× g for 10 min, washed twice with distilled water, and freeze-dried. Approximately 2 mg of freeze-dried microalgal biomass was combined with 1.0 g of micro-beads in a 2 mL tube, and 1 mL of extraction solvent (methanol: dichloromethane: 1:1, v/v) containing 0.025 M NaOH was added. Cells were disrupted using a model FastPrep-24 bead beater (three cycles of 6 m/s for 30 s; MP Biomedicals, Irvine, CA, USA). The microalgal extract was saponified, filtered, and analyzed using an Agilent 1260 infinity device (Hewlett-Packard, Santa Clara, CA, USA) equipped with a diode array detector and a carotenoid column (250 × 4.6 mm, 5 µm; YMC Inc., Devens, MA, USA).

2.6. Other Analytical Methods

Dry cell weight was determined gravimetrically by filtering 4 mL of the H. lacustris broth through a 47 mm GF/C filter (Whatman, Buckinghamshire, UK). The filter was washed with distilled water and dried at 70 °C overnight [43]. pH was measured using a model HM-30R pH meter (DKK-TOA Co., Tokyo, Japan) and light intensity was measured with a model LI-250A quantum photometer (Li-Cor. Inc., Lincoln, NE, USA).

2.7. Statistical Analysis

The experimental data were statistically analyzed using RStudio software (Version 4.2.1.; PBC, Boston, MA, USA) and graphically represented using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA). Statistical significance (p < 0.05) was determined by the analysis of variance using the “agricolae” package, followed by Tukey’s honestly significant difference post hoc test.

3. Results and Discussion

3.1. Life Cycle and Cell Morphology of H. lacustris

Figure 2 illustrates the life cycle of H. lacustris during a 30 d photosynthetic flask cultivation. The figure highlights the three stages of germination, vegetative growth, and encystment; the latter is associated with astaxanthin production. Light microscopy revealed several distinct cell types. Initially, bright red cyst cells from a seed flask culture were inoculated into fresh NIES-C medium. The life cycle begins with cyst germination, where each cyst undergoes multiple internal divisions, producing 2, 4, 8, and up to 16 daughter cells. As germination progresses, the cyst wall ruptures, releasing motile daughter cells.
During vegetative growth, these motile biflagellate cells lose their flagella, enlarge, and transform into spherical green palmella cells rich in chlorophylls referred to as palmella, (I). Under stress conditions, such as nutrient deprivation and aging, H. lacustris palmella cells enter the encystment stage, triggering astaxanthin biosynthesis as a defense against oxidative stress [16,44,45]. These cells then undergo a series of morphological changes, transitioning through green–orange (II), green–brown (III), and reddish (IV) forms, indicating increasing astaxanthin levels. By the end of cultivation, these cells mature into bright red cysts with robust, multilayered cell walls, signifying substantial astaxanthin accumulation [14,46]. However, it is important to note the heterogeneous morphological distribution of cell types during photosynthetic cultivation, with various cell forms evolving over time [47].
This study explored the feasibility of using microfluidic collision treatment to promote cyst germination and enhance astaxanthin productivity in H. lacustris cultures. We varied two key microfluidic parameters—flow rate (1.0–3.0 mL/min) and loop number (3–30)—in a microchannel reactor with T-junction loops to optimize these outcomes.

3.2. Effect of Flow Rate on Seed Cysts and Cell Growth

The impact of varying flow rates from 1.0 to 3.0 mL/min with a fixed loop number of 3 on the viability of H. lacustris cysts is presented in Figure 3a. Cell viability, assessed using trypan blue staining, revealed that untreated cells showed the highest viability at 98.6 ± 1.7%. However, even in controls, some cells might have experienced damage due to shear stress during agitation in the seed flask and subsequent syringe pumping [29].
Cysts exposed to lower flow rates (1.0 to 2.0 mL/min) exhibited viability similar to controls, ranging from 98.1 to 98.7%. However, at higher flow rates (2.5 and 3.0 mL/min), viability slightly decreased to 95.5–95.7%. Microscopic analysis indicated that the cysts treated at lower flow rates maintained morphology similar to controls, while those subjected to higher flow rates showed signs of cellular stress or damage, such as cell wall separation from the cytoplasm and secretion of intracellular materials (Figure S1). These findings suggest that while microfluidic collision can be used to manipulate cysts, precise control of flow rate is critical to maintaining cell viability.
Interestingly, ROS levels, which are typically indicators of cellular stress [48,49], decreased significantly with increasing flow rates from 1.0 to 3.0 mL/min, contrary to expectation (Figure 3b). ROS levels dropped from 322.5 to 200.3, which is notably lower than the control group’s level of 357.4 ± 9.7. This unexpected result might be explained by the possibility that mechanical stress from microfluidic collision induces adaptive responses in the cysts, enhancing their capacity to manage oxidative stress [50]. This hypothesis was further explored (and discussed in subsequent sections) by analyzing changes in astaxanthin content per cell after collision treatment. The reduction in ROS levels suggests that H. lacustris exhibits varied responses to abiotic stress depending on the physiological state of the cell.
This complexity in cellular responses is evident when comparing our results with other studies. For example, Han et al. [38] observed a 14% increase in ROS levels in biflagellate cells of H. lacustris under compressed chip conditions after 6 d of photosynthetic cultivation. Similarly, Sathiyavahisan et al. [43] reported a gradual increase in ROS levels in greenish brown palmella cells of H. lacustris that were electrochemically treated by increasing the current from 5 to 20 mA over 4 h. These varied responses underscore the need for further research to understand the mechanisms underlying stress responses in H. lacustris.
Figure 3c depicts the time course profile of total cell number density of H. lacustris during 72 h of photosynthetic cultivation in a well plate, with cysts treated at microfluidic flow rates ranging from 1.0 to 3.0 mL/min and a fixed loop number of 3. Up to 60 h, the treated cultures showed no significant growth enhancement compared to controls. However, after 72 h of incubation, cultures treated at 2.0 and 2.5 mL/min exhibited higher total cell number densities, reaching 23.5 ± 1.9 × 104 and 21.5 ± 2.2 × 104 cells/mL, respectively, representing increases of 17.5 and 7.6%, respectively, over the control group (20.0 ± 0.9 × 104 cells/mL). These results suggest that microfluidic collision treatment of seed cysts can enhance photosynthetic growth in H. lacustris if carefully controlled, although the effects may appear later than expected. Based on the observed cell density, the optimal flow rate was selected as 2.0 mL/min for subsequent experiments.

3.3. Effect of Loop Number on Seed Cysts and Cell Growth

Figure 4 shows the effect of varying loop numbers (3–30) on H. lacustris cysts at a fixed flow rate of 2.0 mL/min. The loop number correlates with cumulative mechanical stress experienced by the cysts during the microfluidic treatment. As shown in Figure 4a, H. lacustris cysts treated with lower loop numbers (3 and 6) exhibited high viabilities (97.6–97.7%), comparable to the untreated controls (98.1 ± 1.8%). However, increasing the loop number to 10 resulted in a modest reduction in viability to 95.6 ± 0.3%. Further increases to 20–30 loops led to more significant decreases in viability (92.3–94.0%). Notably, at 80 loops, viability dropped sharply to <20%. These findings, combined with findings from varying flow rates (Figure 3a), suggest that while H. lacustris cysts have robust cell walls [6,46], excessive mechanical stress can substantially impair cell viability.
ROS levels in H. lacustris cysts treated with different loop numbers followed a similar trend as with varying flow rates (Figure 4b). All treated cysts exhibited lower ROS levels than controls, with two distinct responses observed. At lower loop numbers (3–20), ROS levels ranged from 241.4–295.5, representing a 24.6–29.8% reduction compared to the control (344.0 ± 6.3). At 30 loops, ROS levels dropped sharply to 193.1, a 44% reduction compared to controls. Microscopic observations revealed significant cell lysis at this loop (Figure S2).
Further analysis focused on the astaxanthin content in H. lacustris cysts after treatment across loop numbers ranging from 3–30 (Figure S3). Notably, higher loop numbers (6–30) resulted in lower astaxanthin content (49.1–53.5 pg/cell) compared to the lowest loop number of 3 and untreated controls (60.1–63.8 pg/cell). This reduction in astaxanthin content suggests that H. lacustris may be utilizing astaxanthin as part of its stress response to mechanical stimulation. Astaxanthin is a potent antioxidant that can be consumed to counteract oxidative stress, indicating a trade-off between stress mitigation and the retention of this valuable carotenoid. Additionally, studies are needed to explore the roles of H. lacustris sophisticated enzyme systems in managing oxidative stress—such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase [45,51]. Understanding these complex biological mechanisms could provide insights into how mechanical stimulation might be harnessed to break the dormancy of microalgal cysts without compromising cell viability.
Figure 4c shows the time course profile of the total cell number density of H. lacustris during 72 h of photoautotrophic cultivation in a well plate, with cysts treated at loop numbers ranging from 3–30 and a fixed flow rate of 2.0 mL/min. Across all loop numbers, treated cultures consistently exhibited higher cell density compared to controls. Cysts treated with an optimal loop number of 10 achieved the highest cell number density after 72 h (28.9 ± 2.0 × 104 cells/mL), representing a 44.5% increase over untreated cells. This significant improvement suggests that microfluidic collision treatment, when carefully controlled, serves as an effective stimulus to enhance the photosynthetic proliferation of H. lacustris cysts.

3.4. Effect of Microfluidic Collision on Cyst Germination Rate

Figure 5a shows the change in the percentage of cysts relative to the total H. lacustris cells during 72 h of photosynthetic cultivation in well plates. The cysts were treated under optimal microfluidic conditions (10 loops at 2.0 mL/min; see Figure 4c). The treated cultures exhibited a significantly faster cyst germination rate between 24 and 60 h compared to the untreated controls. Specifically, after 48 h of incubation, the percentage of cysts in the treated cultures decreased to 18.5 ± 6.1%, whereas the untreated controls maintained a higher cyst percentage of 33.0 ± 5.2%. This accelerated germination process was further supported by the high-speed footage captured from 12 to 72 h for both control and treated cultures (see Supplementary Video S1). We hypothesize that the increased cyst germination efficiency is associated with the enhanced total cell density observed after 72 h, as shown in Figure 4c. This suggests that microfluid collision treatment stimulates the transition from dormant cysts to actively dividing cells and also supports sustained cell growth.
During the cyst germination of H. lacustris, as depicted in Figure 2, a single cyst cell undergoes repeated mitotic events and cytokinesis, resulting in 2n daughter cells (e.g., 2, 4, 8, and 16). To examine the impact of microfluidic treatment on this germination pattern, microscopic images were analyzed at the 48 h mark, considering the significant differences observed between treated and control cultures (Figure 5b). Notably, early germinating stages producing two and four daughter cells were absent, indicating advanced progression in germination events, regardless of microfluidic treatment. Two distinct responses were observed. In untreated cells and those exposed to lower loop numbers (three and six), the proportion of germinating cells producing eight daughter cells was substantially lower, at 56.8–62.5%, compared to the cultures subjected to higher loop numbers (10 to 30), which exhibited ratios of 81.3–81.8%. The data from Figure 5a,b collectively suggest that mechanical collisions influence both the morphology and physiological cell division patterns of H. lacustris cysts.
The increase in cell density may be linked to the activation of mechanoreceptors in the cell wall that induce cell growth [52]. The cell membrane plays a crucial role in various cellular activities, such as activating ion channels and maintaining intracellular ion balance that are vital for cell resistance to mechanical damage. Under mechanical stress, the function, composition, and structure of the cell membrane change to support cell growth and homeostasis [53,54]. For example, Min et al. [35] demonstrated that using a compressed microfluidic culture device for 96 h enhanced S phase and mitosis/cytokinesis of zoospores in C. reinhardtii, resulting in the production of more biflagellate daughter cells compared to uncompressed controls.

3.5. Effect of Microfluidic Collision on Biomass and Astaxanthin Production

To assess the impact of microfluidic collision on microalgal biomass and astaxanthin production, H. lacustris cysts treated under the optimal microfluidic condition (10 loops at 2.0 mL/min, as identified in well plate experiments) were cultured in 120 mL flask setups for 30 d, compared to 2 mL well plates for 3 d, while maintaining consistent photosynthetic conditions across both systems.
Figure 6 shows the time course profiles of total cell number density during photosynthetic flask cultivation. During the initial 3 days, microfluidic collision treatment resulted in a modest increase of 3–10% in cell density compared to the controls. This increase became more significant after 5 d, with treated cultures achieving an 18–30% higher cell density than controls. By the end of the 30 d flask cultivation, the treated H. lacustris cultures reached a total cell density of 32.0 ± 1.1 × 104 cells/mL, an 18.2% increase over the control (27.1 ± 0.3 × 104 cells/mL). While this increase was smaller than the 44.5% observed in the 3 d well plate cultures (Figure 4c), this discrepancy likely reflects differences in photosynthetic culture conditions—such as light transfer, CO2 supply, nutrient mixing, and shear stress—between well plate and flask cultures [55]. Despite these variations, the results from Figure 3c, Figure 4c and Figure 6 consistently demonstrate the positive impact of microfluidic collision treatment on the photosynthetic cell growth of H. lacustris across different culture systems.
Consistent with increased cell density, Figure 7a shows that the final biomass concentration after 30 d in treated H. lacustris cultures (1.48 ± 0.09 g cell/L) was 17.0% higher than in the controls (1.27 ± 0.09 g/L), indicating that enhanced biomass production is a direct result of the improved cell growth facilitated by microfluidic collision treatment. Interestingly, the astaxanthin content per cell remained stable at 19.2–19.4 mg/g cell, regardless of treatment (Figure 7b), suggesting that the mechanical collision of cysts does not directly affect the metabolic pathways for astaxanthin synthesis in H. lacustris. However, due to the increased biomass, astaxanthin productivity in treated cultures (0.95 ± 0.07 mg/L/d) was 21% higher than that in the controls (0.79 ± 0.09 mg/L/d) (Figure 7c). These findings indicate that while mechanical collision treatment enhances overall astaxanthin production by promoting initial germination and subsequent photosynthetic cell growth, it does not alter astaxanthin content per cell.
The biochemical mechanisms and regulation of germination in H. lacustris remain incompletely understood [56]. Previous studies have shown that cyst germination primarily depends on nitrogen levels and light intensity [57]. For example, Choi et al. [19] reported an increase in germination efficiency from 50 to 65% after 4 d when nitrate concentration was raised from 1 to 2 mM under 150 μmol photons/m2/s. Ma et al. [20] highlighted the importance of light wavelengths in cyst germination, observing higher efficiency under blue light compared to white or red light. However, nitrogen supplementation inhibits astaxanthin synthesis, reducing overall astaxanthin productivity [24,27]. Managing light conditions poses challenges due to the need for artificial lighting systems and their associated energy demands [23]. This study is the first to investigate a mechanostimulation strategy for germination in H. lacustris (see next section for detailed discussion). Given the complex life cycle and cellular heterogeneity of H. lacustris (Figure 2 and Figure 5), further research is needed to understand the morphological, biochemical, and genetic changes occurring in treated cysts. Additionally, investigating the kinetics of astaxanthin biosynthesis over time and in relation to cell growth will be a key focus of future studies.

3.6. Process Development for Microalgal Biorefinery

The microfluidic collision treatment presented in this study offers a unique advantage by simultaneously enhancing both biomass and astaxanthin productivity without the need for extreme environmental conditions. By mechanically simulating cyst germination and promoting cell proliferation under ambient conditions, this method avoids the trade-offs typically associated with nutrient starvation [24], high salinity [25], and intensity of light [23]. Moreover, this mechanostimulation strategy is particularly well-suited for integration into biorefinery processes aimed at producing multiple bioactive compounds from H. lacustris. Unlike harsh stress methods that compromise cell viability or require additional chemical inputs [27,30], this approach enhances overall cell growth and biomass accumulation, providing a stronger foundation for extracting other valuable metabolites, such as polyunsaturated fatty acids and carotenoids [7,58]. These attributes make microfluidic collision treatment a promising and environmentally friendly technique for sustainable microalgal biorefinement.
While this study identified optimal flow rates and loop numbers for promoting cell growth, there remains significant potential for further refinement of microfluidic parameters, including cell load, cell alignment efficacy, T-junction design, and channel geometry [40]. Fine-tuning these aspects, along with optimizing photosynthetic culture conditions, such as light intensity, CO2 concentration, and nutrient availability, could further improve astaxanthin productivity [59]. Scaling up the microfluidic collision process for industrial applications will require the development of high-throughput systems capable of efficiently processing large volumes of microalgal cultures [33,60]. Furthermore, exploring the broader application of microfluidic collision treatment beyond H. lacustris could open new opportunities. Given the versatility of this technique, it may be applicable to other microalgal species or even different types of organisms with similar cyst or spore-forming life cycles [61,62,63]. Expanding the scope of this research could establish microfluidic collision as a universal tool for enhancing the productivity of a wide range of bioactive compounds.

4. Conclusions

Mild mechanical collision treatment (10 loops at 2.0 mL/min) using a microfluidic device significantly increased the cyst germination rate of H. lacustris compared to the untreated control. This resulted in a notable enhancement in total cell number density, with a 44.5% increase in well plate cultures over 3 d and an 18.2% increase in flask cultures over 30 d. In flask cultures, treated cultures exhibited a 21% increase in volumetric astaxanthin productivity, primarily due to the higher biomass concentration, while the cellular astaxanthin content of H. lacustris remained unchanged. These results implicate mechanostimulation as a promising and environmentally friendly strategy for enhancing microalgal astaxanthin production and biorefinement, as long as the treatment is carefully optimized to avoid adverse effects on cell viability or function.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app142210434/s1, Supplementary Figure S1: Morphological changes of Haematococcus lacustris cysts after microfluidic collision at different flow rates; Supplementary Figure S2: Morphological changes of Haematococcus lacustris cysts after microfluidic collision at a loop number of 30 and a flow rate of 2.0 mL/min.; Supplementary Figure S3: Astaxanthin content of Haematococcus lacustris cysts treated at different microfluidic loop numbers. Supplementary Video S1: Time-lapse video of the high-speed footage captured from 12 to 72 h for both control and treated cultures.

Author Contributions

Conceptualization, Y.-K.O. and B.K.; methodology, C.C., B.K., A.L.N., L.P.S. and D.P.A.I.; data curation, C.C.; writing—original draft preparation, C.C. and Y.-K.O.; writing—review and editing, Y.-K.O. and E.-J.K.; visualization, C.C., Y.-K.O., A.L.N. and E.-J.K.; supervision, Y.-K.O.; funding acquisition, Y.-K.O. and E.-J.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the STEAM Project (Grant No. RS-2024-00459155) of the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (MSIT). In addition, this work was supported by the Nakdonggang National Institute of Biological Resources (NNIBR; Grant No. NNIBR20243116), funded by the Ministry of Environment (MOE), Republic of Korea.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic diagram of the microfluidic collision treatment applied to Haematococcus lacustris cysts, followed by photosynthetic cultivation in well plates and flasks. The microfluidic chip, inspired by Deng et al. [40], incorporates loops and sharp T-junctions to regulate cell collisions, with adjustable flow rates of 1.0 to 3.0 mL/min and 3–30 loops.
Figure 1. Schematic diagram of the microfluidic collision treatment applied to Haematococcus lacustris cysts, followed by photosynthetic cultivation in well plates and flasks. The microfluidic chip, inspired by Deng et al. [40], incorporates loops and sharp T-junctions to regulate cell collisions, with adjustable flow rates of 1.0 to 3.0 mL/min and 3–30 loops.
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Figure 2. Life cycle of Haematococcus lacustris, illustrating the three distinct cell stages during photosynthetic cultivation: cyst germination, vegetative growth, and encystment. Various cell morphologies, including cysts, germinating cells, released daughter cells, biflagellate cells, and palmella types (I–V), were observed by light microscopy. During germination, a cyst cell undergoes repeated mitotic events and cytokinesis to produce daughter cells, eventually releasing motile cells. In this study, microfluidic collision treatment was applied to H. lacustris cysts to break dormancy and enhance subsequent photosynthetic cell growth.
Figure 2. Life cycle of Haematococcus lacustris, illustrating the three distinct cell stages during photosynthetic cultivation: cyst germination, vegetative growth, and encystment. Various cell morphologies, including cysts, germinating cells, released daughter cells, biflagellate cells, and palmella types (I–V), were observed by light microscopy. During germination, a cyst cell undergoes repeated mitotic events and cytokinesis to produce daughter cells, eventually releasing motile cells. In this study, microfluidic collision treatment was applied to H. lacustris cysts to break dormancy and enhance subsequent photosynthetic cell growth.
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Figure 3. Effect of microfluidic flow rate on (a) cell viability and (b) reactive oxygen species levels in Haematococcus lacustris cysts. (c) Time course profile of total cell number density for 72 h of photosynthetic well plate cultivation using cysts treated at different microfluidic flow rates (1.0, 1.5, 2.0, 2.5, and 3.0 mL/min). Error bars represent the mean and standard deviation of three independent experiments (n = 6). Different italicized letters (a–e) indicate the significance of the differences (p < 0.05). DCF: 2′,7′-dichlorofluorescein. n.s. denotes non-significance between the groups.
Figure 3. Effect of microfluidic flow rate on (a) cell viability and (b) reactive oxygen species levels in Haematococcus lacustris cysts. (c) Time course profile of total cell number density for 72 h of photosynthetic well plate cultivation using cysts treated at different microfluidic flow rates (1.0, 1.5, 2.0, 2.5, and 3.0 mL/min). Error bars represent the mean and standard deviation of three independent experiments (n = 6). Different italicized letters (a–e) indicate the significance of the differences (p < 0.05). DCF: 2′,7′-dichlorofluorescein. n.s. denotes non-significance between the groups.
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Figure 4. Effect of microfluidic loop number on (a) cell viability and (b) reactive oxygen species levels in Haematococcus lacustris cysts. (c) Time course profile of total cell number density for 72 h of photosynthetic well plate cultivation using cysts treated with varying loop numbers (3, 6, 10, 20, and 30). Error bars represent the mean and standard deviation of three independent experiments (n = 6). Different italicized letters (a, b, ab, and c) indicate the significance of the differences (p < 0.05). DCF: 2′,7′-dichlorofluorescein. n.s. denotes non-significance between the groups.
Figure 4. Effect of microfluidic loop number on (a) cell viability and (b) reactive oxygen species levels in Haematococcus lacustris cysts. (c) Time course profile of total cell number density for 72 h of photosynthetic well plate cultivation using cysts treated with varying loop numbers (3, 6, 10, 20, and 30). Error bars represent the mean and standard deviation of three independent experiments (n = 6). Different italicized letters (a, b, ab, and c) indicate the significance of the differences (p < 0.05). DCF: 2′,7′-dichlorofluorescein. n.s. denotes non-significance between the groups.
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Figure 5. (a) Time course profile of the cyst cell ratio relative to total cell count during 72 h of photosynthetic well plate cultivation of Haematococcus lacustris using cysts treated with the optimal microfluidic treatment conditions (10 loops at 2.0 mL/min). (b) Ratio of germinating cell types producing 8 and 16 daughter cells after 48 h of well plate incubation with cysts treated at different microfluidic loop numbers (3, 6, 10, 20, and 30). Refer to Figure 2 for representative microscopic images of germinating cells. Each sample includes at least 100 randomly selected cells from three independent experiments (n = 6). Different italicized letters (a, ab, b, c, d, e, and f) indicate the significance of the differences (p < 0.01). n.s. denotes non-significance between the groups.
Figure 5. (a) Time course profile of the cyst cell ratio relative to total cell count during 72 h of photosynthetic well plate cultivation of Haematococcus lacustris using cysts treated with the optimal microfluidic treatment conditions (10 loops at 2.0 mL/min). (b) Ratio of germinating cell types producing 8 and 16 daughter cells after 48 h of well plate incubation with cysts treated at different microfluidic loop numbers (3, 6, 10, 20, and 30). Refer to Figure 2 for representative microscopic images of germinating cells. Each sample includes at least 100 randomly selected cells from three independent experiments (n = 6). Different italicized letters (a, ab, b, c, d, e, and f) indicate the significance of the differences (p < 0.01). n.s. denotes non-significance between the groups.
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Figure 6. Time course profile of total cell number density during 30 d of photosynthetic flask cultivation of Haematococcus lacustris cysts treated with the optimal microfluidic collision conditions (10 loops at 2.0 mL/min). Error bar represents the standard deviations of three independent experiments (n = 6).
Figure 6. Time course profile of total cell number density during 30 d of photosynthetic flask cultivation of Haematococcus lacustris cysts treated with the optimal microfluidic collision conditions (10 loops at 2.0 mL/min). Error bar represents the standard deviations of three independent experiments (n = 6).
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Figure 7. Comparison of (a) dry cell weight, (b) astaxanthin content, and (c) astaxanthin productivity after 30 d of photosynthetic flask cultivation of Haematococcus lacustris cysts with and without microfluidic collision treatment (10 loops at 2.0 mL/min). Error bars represent the standard deviations of three independent experiments (n = 6). Different italicized letters (a and b) indicate the significance of the differences (p < 0.05). n.s. denotes non-significance between the groups.
Figure 7. Comparison of (a) dry cell weight, (b) astaxanthin content, and (c) astaxanthin productivity after 30 d of photosynthetic flask cultivation of Haematococcus lacustris cysts with and without microfluidic collision treatment (10 loops at 2.0 mL/min). Error bars represent the standard deviations of three independent experiments (n = 6). Different italicized letters (a and b) indicate the significance of the differences (p < 0.05). n.s. denotes non-significance between the groups.
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Christabel, C.; Kim, B.; Lakshmi Narasimhan, A.; Sathiyavahisan, L.P.; Ilhamsyah, D.P.A.; Kim, E.-J.; Oh, Y.-K. Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts. Appl. Sci. 2024, 14, 10434. https://doi.org/10.3390/app142210434

AMA Style

Christabel C, Kim B, Lakshmi Narasimhan A, Sathiyavahisan LP, Ilhamsyah DPA, Kim E-J, Oh Y-K. Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts. Applied Sciences. 2024; 14(22):10434. https://doi.org/10.3390/app142210434

Chicago/Turabian Style

Christabel, Catherine, Bolam Kim, Aditya Lakshmi Narasimhan, Laxmi Priya Sathiyavahisan, Dea Prianka Ayu Ilhamsyah, Eui-Jin Kim, and You-Kwan Oh. 2024. "Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts" Applied Sciences 14, no. 22: 10434. https://doi.org/10.3390/app142210434

APA Style

Christabel, C., Kim, B., Lakshmi Narasimhan, A., Sathiyavahisan, L. P., Ilhamsyah, D. P. A., Kim, E. -J., & Oh, Y. -K. (2024). Enhanced Cell Growth and Astaxanthin Production in Haematococcus lacustris by Mechanostimulation of Seed Cysts. Applied Sciences, 14(22), 10434. https://doi.org/10.3390/app142210434

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