Development and Validation of an Automated Stirred-Tank Photobioreactor for Astaxanthin Production from Haematococcus pluvialis

Abstract

The aim of this study was to design and validate an automated 5 L prototype Stirred-Tank Photobioreactor (ST-PBR) dedicated to the two-stage cultivation of the microalga Haematococcus pluvialis. The classic limitations of stirred-tank reactors (such as high shear stress and suboptimal light penetration) were overcome through precise phase-controlled illumination (60 and 300 μmol m⁻² s⁻¹) and the implementation of an advanced embedded control system integrated with Keysight VEE Pro 9.33 software. The design features an innovative mixing system utilizing a dual marine impeller driven by a brushless motor—operating at a mathematically defined tip speed of 0.48 m/s to preserve cellular integrity—alongside a precise gas dosing strategy (pH-stat) employing medical-grade components. Process verification demonstrated highly stable operation, maintaining a dry biomass concentration of 1.315 g/L with no recorded sedimentation, while achieving a highly competitive astaxanthin biosynthesis yield of 4.12% dry weight (DW). Furthermore, enzymatic extraction facilitated the recovery of a product with high biological activity, as confirmed by an increase in equine adipocyte viability up to 128.1 ± 3.1% in in vitro MTS assays, highlighting its potential for veterinary nutraceutical applications. The developed solution represents a scalable, cost-effective, and viable alternative to advanced tubular photobioreactors.

1. Introduction

Natural astaxanthin, derived from the microalga Haematococcus pluvialis, is one of the most highly sought-after carotenoids in the pharmaceutical and nutraceutical markets [1,2]. This compound commands market prices many times higher than its synthetic counterparts, which necessitates the implementation of advanced bioprocess strategies within the framework of a circular economy [3]. This is driven not only by consumer preferences but, above all, by the significantly higher antioxidant activity, safety of use, and superior bioavailability of the esterified form produced by algal cells [4,5]. However, the industrial production of this metabolite encounters a significant technological barrier arising from the complex life cycle of the microorganism. This process requires cultivation in two radically different physicochemical environments: the proliferation phase (the so-called green, vegetative phase, aimed at biomass accumulation) and the stress phase (induction of carotenogenesis, during which cells transform into red aplanospores) [6,7,8].

The main engineering challenge in H. pluvialis cultivation lies in developing a scalable industrial system that simultaneously provides the gentle conditions required by these fragile cells. Classical solutions, including open systems and traditional closed photobioreactors, require a compromise between mass and gas transfer efficiency and the risk of biomass damage. Vegetative cells, which lack a thick rigid cell wall, are highly susceptible to shear stress generated by mixing and aeration systems [9,10,11]. Furthermore, enclosed bioreactor systems are prone to severe overheating induced by intense illumination, necessitating highly efficient and precise temperature control mechanisms.

The study aimed to design, construct, and test—building upon the authors’ previous experience—a new-generation 5 L Stirred-Tank Photobioreactor (ST-PBR) equipped with a control system based on so-called “Time-dependent Scheduled Task” (TdST) commands. The TdST protocol significantly enhances the long-term stability of the ST-PBR by precisely controlling light excitation intensity, CO₂ flow rate, and temperature regimes. As demonstrated in recent studies on automated, modular cultivation systems, precise online parameter control and automation currently represent a key direction in optimizing bioprocesses for demanding microalgae [12,13,14]. This study hypothesizes that the application of precise impeller torque control and buffer-free pH regulation (via an integrated CO₂ dosing system) will enable the effective adaptation of cost-efficient and scalable ST-PBR technology to the requirements of H. pluvialis, thereby eliminating the historical hydrodynamic limitations of such reactors [15].

The microalga H. pluvialis holds a key position in industrial biotechnology as the richest known source of natural astaxanthin [16,17]. This compound exhibits an antioxidant potential significantly greater than that of β-carotene or vitamin E, which is reflected in the growing market demand within the aquaculture, nutraceutical, and advanced cosmetology sectors [18,19]. A crucial factor driving the direction of current research is the absolute superiority of the natural product over the synthetic one. Chemically synthesized astaxanthin, derived from petrochemical precursors, is characterized by significantly lower bioavailability and biological activity compared to the esterified all-trans form produced by algae. For this reason, the optimization of photobioreactor (PBR) systems for H. pluvialis currently represents a priority in bioprocess engineering [1,20,21]. Microalgal biomass production is currently carried out in open systems and closed photobioreactors (PBRs), each possessing specific engineering characteristics [3,22,23,24]. While open ponds [25,26] and closed designs such as tubular reactors [27,28] or airlift columns [29,30] offer distinct advantages for specific microalgae, ST-PBRs represent a direct adaptation of classical fermenters for phototrophic processes.

Although they constitute a standard in industrial biotechnology, ST-PBRs are less frequently applied in H. pluvialis cultivation due to specific hydrodynamic challenges. Despite the dominant position of these reactors in the fermentation industry, their historical application has been limited by two critical factors: shear stress and light distribution. Traditional impellers (e.g., Rushton turbines) generate high velocity gradients, leading to mechanical damage of fragile vegetative cells as observed in various microalgae [31], or the inhibition of encystment specifically in H. pluvialis [32]. Moreover, in classical steel tanks at high culture optical densities, light limitation occurs in the center of the reactor (the so-called dark zone), which necessitates the use of advanced internal illumination systems [33,34]. In the presented solution, the decision was made to base the research platform on a modified ST-PBR geometry to overcome these limitations through a fully programmable system capable of multi-parameter control and virtualization of algal cultivation. A complete schematic diagram of the designed system, encompassing sensor arrays and actuators, is presented in Figure 1.

The key design premises included industrial scalability, platform versatility, maximization of bioprocess yield, and validation by a Stress-Test scenario. The selection of an industry-standard geometry eliminates implementation barriers, allowing a process developed in a 5 L ST-PBR to be effectively transferred to multi-ton installations by maintaining the principles of geometric and kinematic similarity [35,36,37]. The ST-PBR construction enables a flexible shift in the production profile (e.g., to heterotrophic bacterial fermentation) by modifying control parameters without the need for hardware reconfiguration [23,38]. To achieve a high biomass concentration without inducing premature cellular stress [12], the application of the ST-PBR aimed to drastically enhance growth kinetics. The objective was to demonstrate that, through hydrodynamic optimization and precise CO₂ dosing [27], a stirred-tank reactor can match or even surpass the performance of traditional tubular photobioreactors, achieving maximum optical density prior to the induction of carotenogenesis [30]. The utilization of the shear-sensitive microalga H. pluvialis was treated as the ultimate verification of the system’s precision, hypothesizing that high cell viability would serve as proof of the efficacy of the proprietary low-shear impeller and the advanced control system [39,40].

The main motivation of this study was to design, automate, and evaluate a scalable ST-PBR system capable of overcoming typical shear-stress limitations. By implementing a precise pH-stat CO₂ feeding strategy and optimized light regimens, this proof-of-concept validation aims to demonstrate that a fully automated ST-PBR can provide a highly competitive environment for both biomass proliferation and high-yield bioactive astaxanthin accumulation in a sequential two-stage batch process.

2. Materials and Methods

2.1. Haematococcus pluvialis and Cultivation Conditions

The Haematococcus pluvialis G1002 microalgal strain, obtained from the Culture Collection of Algae of Charles University in Prague (CAUP), was used in this study. Morphological observations and original photographic documentation of the cells (Figure 2) were performed in brightfield mode using a Leica DM1000 optical microscope (Leica Microsystems, Wetzlar, Germany) equipped with a dedicated high-resolution digital camera. Image analysis was conducted at 400× magnification. Pre-cultures (inocula) were maintained in sterile 1 L laboratory bottles containing Bold’s Basal Medium (BBM; Sigma-Aldrich, St. Louis, MO, USA) [23,41]. The cultures were incubated at 22 ± 1 °C under a 12:12 h light/dark (L/D) photoperiod. The initial pH of the medium was adjusted to 7.3, ensuring optimal conditions for vegetative growth [22,42].

2.2. Construction and Mixing System

The TdST research platform for the microalgal cultivation process was based on an ST-PBR vessel with a working volume of 5 L. Both the cylindrical tank and the reactor headplate were fabricated from high-quality 3.3 borosilicate glass, ensuring maximum light transmission within the photosynthetically active radiation (PAR) range and providing resistance to sterilization [35]. The interface between the headplate and the vessel was sealed with a silicone O-ring and secured with a quick-release clamp, guaranteeing the hermetic integrity of the system. The vessel was equipped with an integrated water jacket functioning as a heat exchanger (Figure 3a). The deionized water utilized within the jacket also served as an optical filter, absorbing infrared (IR) radiation in the bioreactor’s illumination path.

The mixing system is driven by a Teknic brushless motor controlled by an Aerotech BA20 (Aerotech Inc., Pittsburgh, PA, USA) in a constant velocity control mode using quadrature encoder feedback. Torque transmission is achieved using an AISI 316L stainless steel shaft featuring a dual marine-type propeller system. The lower impeller was positioned just above the bottom of the vessel to prevent cyst sedimentation, while the upper one was located at the mid-height of the liquid column, ensuring efficient axial circulation [36] (Figure 3c). An unbaffled vessel geometry was applied, which, combined with the polishing of metal components (Ra < 0.4 µm), drastically reduces high-shear-stress zones [31,38]. Agitation in the ST-PBR was provided by a proprietary low-shear impeller operating at a constant speed of 120 RPM. This configuration generated a maximum tip speed of 0.48 m/s, which was experimentally determined to ensure adequate mixing while remaining below the critical shear stress threshold for vegetative H. pluvialis cells.

2.3. Lighting and Thermoregulation System

Due to the phototrophic requirements of H. pluvialis, the bioreactor design was based on an external, modular, 4-channel 630 nm LED lighting system. The modular light panels, mounted on aluminum heatsinks around the water jacket, emit light with a spectrum tailored for the absorption of photosynthetic pigments [15] (Figure 3c). Illumination was supplied by an LED array providing a precisely controlled Photosynthetic Photon Flux Density (PPFD). During the initial green vegetative phase, the PPFD was maintained at 60 μmol m⁻² s⁻¹ to promote optimal biomass proliferation. Upon initiation of the stress phase (carotenogenesis), the light intensity was increased to 300 μmol m⁻² s⁻¹ to effectively induce astaxanthin accumulation.

Distilled water was chosen as the medium for the temperature regulation of the ST-PBR. The main control unit is based on a precision Azbil SDC35 PID controller, operating within a temperature range of 18.00 °C to 32.00 °C. Continuous linear regulation is achieved using bridged Apex PA26 power operational amplifiers (Apex Microtechnology, Tucson, AZ, USA) that supply Peltier modules mounted in a frame manufactured by Roche. During the culture heating phase (24.00–30.00 °C), an additional resistance heater mounted on a quartz tube was employed, thereby shortening the heating time and improving the dynamics of temperature regulation. The temperature controller compensates for the Joule heating generated by the LED lighting, maintaining the process temperature with an accuracy of ±0.02 °C [39] (Figure 3a).

2.4. CO₂ Dosing System

To eliminate fluctuations in carbon dioxide delivery, a dosing system based on medical-grade pressure regulators was designed (Figure 3c). This choice ensures the stability of the CO₂ partial pressure, which is critical for the process [43]. The gas lines were equipped with 0.22 µm PTFE membrane filters to ensure the sterility of the inlet medium. CO₂ is introduced into the reactor through a microporous sparger located beneath the lower impeller, ensuring the immediate dispersion of bubbles. Aeration and precise pH control were maintained simultaneously via a pH-stat strategy, utilizing a CO₂-enriched air stream with a total gas flow rate of 0.1 vvm.

2.5. Control System

The bioprocess is supervised by an integrated automation system operating within the Keysight VEE Pro 9.33 environment (Keysight Technologies, Santa Rosa, CA, USA). The VEE platform acquires real-time signals from all sensors: pH, temperature, gas concentration, and light intensity [9]. The software enables the execution of TdST commands, including the automated adjustment of control parameters in response to the culture’s growth dynamics. The entire station was enclosed within a dedicated protective frame featuring an inspection window, ensuring the mechanical stability of the glass vessel and operator safety (Figure 3b).

2.6. Analytical Methods

2.6.1. Biomass Growth Determination

Routine monitoring of microalgal growth and physiological state was conducted using a UV-Vis spectrophotometer (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). Optical density was measured at two distinct wavelengths to capture different culture dynamics: 680 nm (OD 680 nm) to estimate chlorophyll absorption and monitor active photosynthesis during the vegetative phase, and 750 nm (OD 750 nm) to evaluate total cell turbidity and biomass concentration independently of pigment profile shifts. To avoid sampling errors caused by the rapid sedimentation of encysted cells during the red phase, 20 mL aliquots were collected under vigorous continuous mixing.

Additionally, absolute biomass growth was monitored using a gravimetric method. Culture samples of 20 mL were collected in triplicate and centrifuged (4000 rpm, 10 min) to separate the biomass from the culture medium. The supernatant was discarded, and the cell pellet was dried under a nitrogen stream until a constant dry weight was achieved, after which it was weighed on an analytical balance.

2.6.2. Extraction and Enzymatic Hydrolysis of Astaxanthin Esters

Sample preparation for high-performance liquid chromatography (HPLC) analysis was conducted according to a modified protocol described by Wang et al. [44,45], comprising dimethyl sulfoxide (DMSO) extraction and enzymatic hydrolysis. One milliliter of DMSO was added to the cell pellet (5–20 mg), and the mixture was incubated at 60 °C for 5 min to permeabilize the cell wall. Subsequently, the samples were centrifuged (9600× g, 2 min), and the supernatant was collected. The remaining pellet was extracted repeatedly with acetone until the complete decolorization of the biomass. The collected fractions (DMSO and acetone) were combined.

To release free astaxanthin from its esterified forms, enzymatic hydrolysis was performed using cholesterol esterase. Tris-HCl buffer (pH 7.0) and the enzyme solution were added to the extract, and the mixture was incubated in a water bath at 37 °C for 30 min [46]. The liberated carotenoids were extracted in triplicate with petroleum ether, which was subsequently evaporated to dryness under a nitrogen stream. The dry residue was dissolved in 1 mL of an acetone–methanol mixture (1:1, v/v) and subjected to HPLC analysis.

2.6.3. HPLC Analysis

Chromatographic separation was performed using an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Luna C18 column (150 × 4.6 mm, 300 Å; Phenomenex, Torrance, CA, USA). UV-Vis detection was carried out at a wavelength of 474 nm. An isocratic system consisting of methanol and water in a 92.5:7.5 (v/v) ratio was used as the mobile phase. The mobile phase flow rate was 1.0 mL/min, the column temperature was maintained at 30 °C, and the injection volume was 10 µL. The identification and quantification of astaxanthin were based on an external standard calibration curve (Sigma-Aldrich, St. Louis, MO, USA) [47].

2.6.4. Bioactivity Assessment

The impact of the extracts on cell viability was evaluated using the colorimetric MTS tetrazolium salt reduction assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) on an equine adipocyte cell line. The extracts were prepared analogously to Section 2.6.2; however, the final solvent (acetone-methanol) was evaporated, and the dry residue was resuspended in the culture medium supplemented with DMSO (final concentration < 0.1%) to eliminate the toxicity of organic solvents. The cells were incubated with the tested extracts for 24 h. Absorbance was measured at a wavelength of 490 nm, and the results were compared to the control group [48,49].

2.6.5. Statistical Analysis

To ensure the reliability and reproducibility of the bioprocess evaluation, the 34-day ST-PBR cultivations were performed in independent biological triplicates (n = 3). All subsequent analyses were also performed in three independent replicates. The results are expressed as the arithmetic mean ± standard deviation (SD) [50].

3. Results

3.1. Bioreactor Performance Characteristics and Process Stability

The prototype ST-PBR system was verified under continuous operation conditions. A key engineering challenge was the stabilization of the biomass suspension in the red phase, where heavier aplanospores exhibit a tendency to sediment. The applied dual-impeller configuration ensured effective axial circulation and medium homogenization throughout the 5 L volume, preventing biomass from settling at the bottom of the vessel. The thermoregulation system (Peltier/water jacket) effectively compensated for the heat generated by the LED lighting, maintaining the process temperature at a set point of 22.00 ± 0.02 °C. Notably, due to the electropolishing of steel components and the use of borosilicate glass, the phenomenon of biomass adhesion (biofouling) on the reactor walls was minimized. The gas dosing system, based on medical-grade regulators, ensured stable CO₂ partial pressure, which translated into precise pH control without the need for aggressive chemical adjusters.

3.2. Growth Kinetics and Phase Transition

To precisely evaluate the culture dynamics and the transition between the vegetative and stress phases, the cultivation process was continuously monitored using spectrophotometric measurements at 680 nm and 750 nm (Figure 4). During the initial exponential growth phase (days 1–8), a rapid and parallel increase in both parameters was observed, culminating on day 14 with a maximum OD 680 of 0.358. This indicates intense chlorophyll synthesis and active cell proliferation. Following the induction of the stress phase (carotenogenesis), the culture exhibited a distinct physiological shift. While the OD 680 plateaued (remaining within the range of 0.351–0.355 between days 14 and 28), reflecting the cessation of chlorophyll synthesis, the total cell turbidity measured at OD 750 continued to rise significantly (from 0.196 to 0.348). This dynamic perfectly illustrates the encystment process: the cells ceased division and redirected their metabolism toward cell wall thickening and the intracellular accumulation of secondary metabolites (astaxanthin, lipids, and carbohydrates), thereby increasing total light scattering without increasing the green pigment content. The subsequent decline in both OD values in the final days of the process (days 28–34) reflects the natural tendency of mature, heavy red cysts to partially sediment or form localized aggregations in the late stages of cultivation, despite continuous agitation.

3.3. Biomass Production Yield and Metabolic Profile

Under photochemical stress conditions induced within the bioreactor, the H. pluvialis G1002 culture demonstrated high morphological transformation efficiency, resulting in a systematic increase in biomass (

Table 1. Measurements of dry biomass accumulation at successive stages of the 34-day cultivation process in the ST-PBR photobioreactor (values for 20 mL sample volumes).

Cultivation Day	Cultivation Stage	Dry Biomass Weight (20 mL Sample) [g]
Day 1	Start	0.0029 ± 0.0002
Day 8	Exponential growth	0.0175 ± 0.0011
Day 14	Max. green phase	0.0241 ± 0.0010
Day 21	Stress induction/Onset of adhesion	0.0249 ± 0.0013
Day 28	Red phase	0.0256 ± 0.0015
Day 34	Final harvest	0.0263 ± 0.0011

). Microscopic observations confirmed that the vast majority of the population transformed into thick-walled, red cysts, indicating uniform light availability throughout the reaction vessel volume. Based on the measurements conducted, the final dry biomass weight (final harvest on day 34) was 0.0263 g in a 20 mL sample, which corresponds to a concentration of 1.315 g/L when extrapolated to the reactor’s working volume.

HPLC analysis, preceded by enzymatic hydrolysis, revealed the presence of astaxanthin as the dominant carotenoid. This is illustrated by the obtained chromatogram (Figure 5), which is characterized by a very clean baseline and a prominent peak with a retention time of approximately 5.0 min, with a negligible contribution of signals from other pigments. The final content of this compound reached 4.12% ± 0.15% of the dry weight (DW). This result confirms that the designed control system effectively induced the secondary metabolite biosynthesis pathway while preserving cellular integrity through the minimization of shear forces.

3.4. Bioactivity and Cytotoxicity

To evaluate product safety, a study was conducted on an equine adipocyte cell line. MTS assay analysis revealed a complete absence of cytotoxic effects from the extract within the tested concentration range (25–100 µg/mL). Furthermore, a dose-dependent, significant stimulation of cellular metabolic activity was observed, as illustrated in the obtained graph (Figure 6). The application of four concentration variants (including the control group) allowed for the determination of a complete biological response curve.

At a dose of 25 µg/mL, a moderate increase in viability to 110.3 ± 2.2% (p < 0.01) was recorded, followed by a rise to 124.8 ± 2.4% (p < 0.001) at a concentration of 50 µg/mL. Conversely, doubling the highest dose (from 50 to 100 µg/mL) resulted in only a marginal increase to 128.1 ± 3.1% (p < 0.001) relative to the control group. This distribution of results indicates the achievement of a saturation point (plateau), where a further increase in extract concentration no longer translates into a proportional increase in metabolic activity. The obtained data suggest that the recovered astaxanthin retained high biological activity, thereby confirming the efficacy and gentle nature of the applied production process and enzymatic extraction.

4. Discussion

4.1. Bioprocess Efficiency in the Context of Advanced Control

The final astaxanthin content of 4.12% dry weight (DW) achieved in this study is a highly satisfactory result, placing the prototype system in the upper tier of yields reported for phototrophic technologies [1,8,51]. The literature indicates that in standard cultivation systems, the average carotenoid accumulation typically ranges within lower brackets (1.5–3%) [10,11,52]. Exceeding the 4% threshold in an ST-PBR demonstrates that the precise control of environmental parameters can overcome the natural biological limitations of the strain. This result substantiates the thesis that the key to maximizing yield is not solely the reactor geometry, but the advanced integration of control subsystems [4,5,53]. In the presented prototype, the strict control of light stress and nutrient availability allowed for the maintenance of optimal induction conditions without the risk of culture collapse, which is crucial for sustainable biomass production [26].

It should be noted that the final biomass concentration of 1.315 g/L achieved in this study is somewhat lower compared to highly optimized closed tubular photobioreactors [54]. Nevertheless, the presented ST-PBR system offers a range of undeniable technological and economic advantages that compensate for this difference. The prototype was intentionally designed using cost-effective components and drive solutions adapted from other engineering fields, which drastically reduces initial capital expenditures (CAPEX). Furthermore, tank-type ST-PBRs are characterized by incomparably simpler operation, including ease of cleaning, sterilization, and maintenance, relative to complex tubular systems. Crucially, while the volumetric biomass yield may be lower, the intracellular astaxanthin accumulation reached a highly competitive 4.12% of dry weight (DW). This clearly demonstrates that the ST-PBR, through efficient mixing and light distribution, creates optimal stress conditions that maximize the biosynthesis of the high-value target product, making it a highly pragmatic and scalable alternative for commercial astaxanthin production.

4.2. Solving the Hydrodynamics Problem in ST-PBRs

ST-PBRs are frequently considered less suitable for the cultivation of sensitive microalgae due to the generation of high shear forces by mixing systems [9,14]. The present study challenges this view, demonstrating that proper engineering of the mixing system allows for the effective utilization of this technology [17]. While advanced fluid flow mapping via computational fluid dynamics (CFD) would provide deeper mechanistic insights into shear distribution [55], our empirical approach—maintaining a maximum tip speed of 0.48 m/s—was highly effective. The application of a dual-impeller configuration with marine-type propellers and a servo drive enabled the generation of a circulatory, rather than turbulent, flow profile. This solution effectively prevented the sedimentation of heavy cysts—a common technological bottleneck—while simultaneously preserving full cellular integrity [18]. By relying on established historical baselines for standard impellers rather than conducting parallel runs with unoptimized conventional reactors, this proof-of-concept focused exclusively on validating the improved dual-impeller architecture. The absence of foam and biofouling on internal components further validates the adopted engineering strategy [56].

4.3. Innovation in the Gas Dosing System

A unique aspect of the presented solution is the adaptation of medical-grade components for gas dosing control, representing a significant upgrade over standard laboratory setups. This system ensured exceptional stability of the CO₂ partial pressure, which, in the context of Industry 4.0 trends and the “Smart Bioreactor” concept, constitutes a major conceptual advantage [4,27,57]. The implemented solution enabled the precise execution of a pH-stat strategy without the need for aggressive chemical buffers. Stabilizing the pH solely through gas flow modulation is a “clean” method that does not introduce additional osmotic stress to the medium, directly translating into the high physiological condition of the biomass and the absence of contamination [20,58].

4.4. Product Bioactivity and the Extraction Method

The high result of the MTS assay (128.1% cell viability) indicates the excellent biological quality of the obtained product. Many commercial astaxanthin production methods rely on aggressive chemical extraction, which can diminish the compound’s antioxidant potential [23,59]. The obtained results suggest that combining mild cultivation conditions in the bioreactor (absence of overheating due to Peltier cells) with the enzymatic hydrolysis method allowed for the preservation of the native structure of the astaxanthin molecules [21,60].

The selection of equine adipocytes as an in vitro cell model for evaluating the bioactivity of the extracted astaxanthin was driven by physiological relevance and practical application. First, this choice directly aligns with the targeted commercial application of the studied microalgal extracts, which are being developed as innovative dietary supplements and veterinary preparations for horses (e.g., to mitigate oxidative stress in performance animals). Furthermore, since astaxanthin is a highly lipophilic compound that naturally accumulates in lipid-rich tissues, utilizing homologous fat cells from the target species provides a highly relevant and physiologically accurate model for assessing its in vivo biological potential. Second, this specific cell line constituted a well-characterized, stable, and highly reproducible in vitro model in our laboratory. Utilizing a validated cellular model with known growth kinetics ensured a rigorous and artifact-free evaluation of the cytotoxicity and biological activity of the recovered carotenoids. The proliferative effect observed during in vitro studies confirms that the designed process yields a highly pure and biologically active raw material, safe for mammalian cells [25,61,62,63]. Future studies should incorporate a pure astaxanthin standard as a positive control to definitively attribute the observed proliferative effects solely to astaxanthin, rather than potential synergistic effects of the total extract matrix.

5. Conclusions

Based on the conducted structural analyses and process studies, it can be concluded that the primary objective of this work—the design, optimization, and commissioning of an automated photobioreactor for the efficient cultivation of H. pluvialis—has been fully achieved. A solution comparable to expensive products from renowned manufacturers (e.g., IKA) was developed, featuring a proprietary control system and TdST software (version 1.0). The following conclusions were drawn:

• Validation of the ST-PBR concept for sensitive cultures: It was demonstrated that appropriate modification of the mixing system (application of a dual marine-type impeller and a brushless drive with precise torque control) allows for the efficient cultivation of H. pluvialis in a stirred-tank reactor. This dispels the common belief regarding the unsuitability of classical ST-PBRs for algal bioprocesses due to shear forces.
• Biosynthesis yield: The integrated system for controlling stress parameters (light/nutrients) resulted in an astaxanthin yield of 4.12% DW. This result surpasses the standard performance of open systems and is competitive with advanced tubular photobioreactors, while maintaining lower investment costs.
• Innovation in pH control: Replacing chemical buffers with precise CO₂ dosing (using medical regulators) enabled residue-free pH stabilization. This strategy not only simplifies process operation but also eliminates the risk of product contamination by salts formed during acid-base neutralization.
• Product quality: The application of mild processing conditions and enzymatic extraction preserved the high biological activity of astaxanthin. The increase in cell viability to 128.1% in the MTS assay proves that the obtained product possesses proliferative potential and is free from toxic degradation products.
• Implementation potential: Through the application of industrial standards, the developed prototype constitutes a viable research platform, scalable to pilot volumes. It aligns with Industry 4.0 requirements through its capacity for multiparameter virtualization and automated cultivation control, facilitating future scale-up to industrial conditions. Furthermore, future research will focus on a direct, parallel comparative analysis between the developed ST-PBR and an air-lift photobioreactor to comprehensively evaluate their respective hydrodynamic, energetic, and mass transfer efficiencies.