Effect of Photoperiod Duration and LED Light Quality on the Metabolite Profiles of High-Mountain Microalgal Isolates

Abstract

High-mountain microalgae exhibit remarkable adaptability to extreme environments, making them promising candidates for sustainable biorefineries. We evaluated how photoperiod (12:12, 18:6, 24:0 h) and LED spectra (cool white, full spectrum, red–blue 4:1) affect growth and metabolite formation in Chlorella sp. UFPS019 and Scenedesmus sp. UFPS021. Biomass peaked in Chlorella under red–blue 18:6 (≈1.8 g L⁻¹) and in Scenedesmus under red–blue 24:0 (≈1.7 g L⁻¹), revealing species-specific responses. Carbohydrate fractions were maximized under red–blue 12:12 in both species, and continuous light (24:0) depressed carbohydrate content—most notably under full spectrum. Protein content was highest under red–blue 18:6 in Chlorella sp. and under red–blue 12:12–18:6 in Scenedesmus sp. Lipid fractions increased with light duration, peaking under red–blue 18:6–24:0 in Chlorella and under red–blue 18:6–24:0—with Cool White 24:0 also high—in Scenedesmus sp. Although extended illumination favored lipids, intermediate photoperiods (12:12–18:6) provided better productivity-to-energy trade-offs and broader metabolic profiles. These results show that tuning spectral composition and photoperiod to species-specific physiology enables the targeted, energy-aware production of proteins, carbohydrates, or lipids; red–blue at intermediate durations is a robust, energy-efficient regime, whereas longer exposures can be used strategically when lipid enrichment is prioritized.

1. Introduction

Microalgae and cyanobacteria are photoautotrophic microorganisms widely distributed in freshwater and marine environments, exhibiting remarkable physiological and biotechnological versatility, which enables their integration into bioproduct platforms of industrial interest [1]. The incorporation of these platforms into circular economy schemes relies on the capacity of microalgae to convert residual nutrients into useful biomass. Experimental evidence indicates that adjusting carbon sources and nitrogen–phosphorus (N–P) ratios in aquaculture effluents increases biomass yield and improves product quality [2,3,4]. Nevertheless, recent reviews converge on the conclusion that production costs and bottlenecks in biomass recovery and stabilization continue to limit large-scale adoption, leading to a prioritization of cultivation intensification strategies and precise control of abiotic variables [5].

Among these variables, light plays a central role due to its ability to simultaneously modulate growth and carbon allocation into value-added fractions—carbohydrates, lipids, and proteins—across different genera [6]. Photoperiod duration, effective intensity, and spectral quality function as signals that regulate primary metabolism and storage pathways; for example, intermediate day–night regimes have demonstrated biomass gains by balancing photosynthetic activity with dark-phase repair processes [7]. The adoption of light-emitting diodes (LEDs) in closed photobioreactors has gained prominence, owing to their electrical efficiency, stability, and capacity for precise spectral programming, thereby enabling metabolic targeting strategies toward proteins or oils depending on process objectives. Several recent analyses document this approach and its relevance for industrial scale-up [8,9]. The light effectively perceived by the cells further depends on hydrodynamics and radiative transport within the culture. Studies that minimized self-shading and decoupled mixing from illumination confirmed growth improvements when temporal scales of light/dark exposure were controlled [7]. Pilot-scale trials with Chlorella and Scenedesmus have employed a 12:12 photoperiod in optimization stages using flat-panel reactors to balance photosynthesis and repair [6], highlighting that temporal regimes and spectral composition must be jointly conceived when designing operational windows for algal bioprocesses. This synchronization with the inherent light–dark rhythms of the day–night cycle is particularly relevant in high-mountain isolates, whose history of exposure to extreme regimes anticipates differentiated photophysiological responses.

High-Andean isolates represent a strategic bioprospecting opportunity due to their evolutionary history under low temperatures, oligotrophy, and high irradiance/UV exposure at altitudes above 3300 m.a.s.l. (meters above sea level). These selective pressures promote photoprotective pigments and efficient antioxidant systems, traits that can translate into productivity and robustness under controlled illumination. In Andean genera such as Pediastrum sp. (>4400 m.a.s.l.), the accumulation of pigments and phenolic compounds with high potency against visible/UV radiation has been reported, favoring tolerance to light stress [10]. Similarly, native high-mountain strains maintain stable levels of chlorophylls and antioxidant pigments and enhance their radical-scavenging capacity under UV-B without compromising photosynthesis [11]. These capabilities—tolerance to high irradiance, efficiency in redox management, and photophysiological plasticity—position high-Andean isolates as suitable chassis for generating fractions of interest (functional proteins, fermentable sugars, and oils) in algal biorefineries. Nevertheless, their response surfaces under LED matrices of photoperiod and spectrum remain poorly characterized compared with lowland model species, particularly when aiming to define operational windows transferable to larger-scale systems. This knowledge gap justifies addressing, under process-based criteria, how spectral programming and photoperiod condition metabolic outputs in organisms naturally exposed to extreme light regimes.

Technological interest in programmable light systems aligns with a bioprocess agenda that prioritizes metabolite-oriented configurations and stream co-processing. The recent literature shows that response models of Chlorella sp. to different spectra position red–blue combinations as levers to maximize growth and proteins, while white-light or broadened-spectrum profiles favor pigments and, under specific phases, lipids. These patterns have been quantified through multivariate analyses and machine learning, reinforcing the role of the spectrum as a process design variable [12]. In parallel, evaluations of the LED-based cultivation of microalgae and cyanobacteria discuss energy efficiency and operational stability as advantages over conventional light sources, in addition to the possibility of modulating cycles and spectral bandwidth to direct carbon partitioning, as documented in recent syntheses [8,12]. Within this framework, the selection of contrasting spectra not only discriminates physiological responses but also guides the bioprospecting of priority metabolites under transferable operational windows.

On this basis, previous studies with the same strains examined in the present work—Chlorella sp. UFPS019 and Scenedesmus sp. UFPS021—established non-restrictive nutritional limits and metabolic profiles under optimized C/N/P (carbon/nitrogen/phosphorus) ratios [13]. This provides a reference point for now interrogating light as the main control factor in carbon allocation. Building on this premise and the accumulated evidence on metabolic plasticity in response to light stimuli, this study hypothesizes that intermediate photoperiod regimes combined with mixed spectra can balance photosynthetic efficiency with cellular recovery processes, thereby maximizing yield without compromising physiological viability. Accordingly, the present investigation characterizes the physiological and biochemical responses of two isolates from the Santurbán páramo (UFPS019 and UFPS021) under programmable LED illumination combining photoperiod durations and spectral compositions (cool white, full spectrum, and red–blue 4:1). The objective is to quantify species-specific growth responses and map the biochemical outputs of direct biorefinery interest—carbohydrates, proteins, lipids, and carotenoids—under light conditions that are controllable and transferable to process scale.

2. Materials and Methods

2.1. Strains

Chlorella sp. (CHLO_UFPS019) and Scenedesmus sp. (SCEN_UFPS021) were previously isolated from high-mountain lakes (3300–3900 M.A.S.L.) within the paramo of the Santurban region, which represents a wide range of conditions that harbor a rich variety of microorganisms in the region of Norte de Santander (Colombia) [12]. The strains were maintained at the INValgae collection (Universidad Francisco de Paula Santander, Colombia) and cultured in a 2 L tubular glass flask with 1.3 L of bold basal medium (BBM) [14]. Each flask was mixed through the injection of filtered air with 1% (v/v) CO₂ at a flow rate of 0.78 L_air/min, with a photoperiod of 12:12 h at 100 µmol m⁻² s⁻¹ for 30 days. Cultures were checked for bacterial background by plating samples on LB agar (no detectable CFU) and by DAPI microscopy.

2.2. Effects of LED Wavelength and Photoperiod

The increases in total carbohydrates, total proteins, and total lipids were evaluated via three different configurations of photoperiods (light–dark, 12:12; 18:6; 24:0) and three different LED sources (cool white LEDs (control) (60 LEDs/m, 400–700 nm, 12 V, 8 W/m) (Sinowell, Shanghai, China), red–blue LEDs (4:1 ratio; 60 LEDs/m, Blue: 660 nm, Red 450 nm, 12 V, 8 W/m) (Sinowell, Shanghai, China), and full spectrum LEDs (60 LEDs/m, 380–780 nm, peak emissions at 450 nm and 660 nm, 12 V, 8 W/m) (Sinowell, Shanghai, China) [15]. For each experiment, the strains were cultured (in triplicate) in 500 mL flasks with a working volume of 250 mL of bold basal medium. Each flask was mixed with filtered air at a flow rate of 0.15 L_air/min at 100 µmol m⁻² s⁻¹ for 20 days for carbohydrates and proteins and 40 days for lipids according to previous results [13].

2.3. Biomass and Metabolite Quantification

The biomass produced was concentrated via centrifugation on a ROTINA 420 R (2054× g, 20 °C, 20 min) (Hettich, Tuttlingen, Germany). The concentrated biomass was lyophilized and stored (4 °C) until use. The final concentration of biomass was recorded as the total produced biomass (g/L).

Total carbohydrates were measured according to Moheimani et al. [16]. Briefly, 5 mg of dried biomass was mixed with 5 mL of 1 M H₂SO₄ in test tubes. The sample was vortexed (Multi Reax, Heidoplh, Schwabach, Germany) at 1500 rpm (10 min) and incubated (100 °C, 60 min). The mixture was cooled down at room temperature and centrifuged (2876× g, 20 min). Two milliliters of the supernatant were mixed with 1 mL of phenol solution (5% w/v) and 5 mL of concentrated H₂SO₄ and cooled down at room temperature. The sample was measured at 485 nm.

Total lipids were measured using the method described by Mishra et al. [17]. Briefly, 5 mg of dried biomass was mixed with 2 mL of concentrated H₂SO₄ and vortexed (Multi Reax, Heidoplh, Schwabach, Germany) at 1500 rpm for 5 min. The sample was then incubated (100 °C, 10 min) and cooled down in an ice bath for 5 min. Five milliliters of fresh phospho-vanillin reagent was added to the sample and it was incubated (37 °C, 15 min). The final sample was centrifuged (2876× g, 20 min), the supernatant was measured at 530 nm.

Finally, the total content of proteins was measured using the method modified by Slocombe et al. [18]. Briefly, 5 mg of dried biomass was vortexed (Multi Reax, Heidoplh, Schwabach, Germany) (1500 rpm, 5 min) with 200 µL of 24% (w/v) trichloroacetic acid (TCA). The mixture was incubated (95 °C, 15 min) and then diluted with 600 µL of ultrapure water. The samples were then centrifuged (15,000× g, 20 min, 4 °C), and the supernatants discarded. The pellets obtained were resuspended in 0.5 mL of Lowry Reagent D and incubated at 55 °C for 3 h. Samples were centrifuged again (15,000× g, 20 min, 4 °C), and the supernatants containing solubilized protein were collected for quantification.

2.4. Statistical Analysis

The results obtained from the quantification of the different metabolites were analyzed via two-way ANOVA (factors: photoperiod and spectrum) in GraphPad Prism v10.5.0. Assumptions of normality and homoscedasticity were verified; Tukey’s HSD was used for multiple comparisons (α = 0.05). Bars depict mean ± SD (biological n = 3). Different letters indicate distinct Tukey groups.

3. Results

First, when biomass concentration was analyzed (Figure 1a), a marked interaction between the evaluated factors was observed. The combination of red–blue light (4:1) under an 18:6 h photoperiod yielded the highest biomass concentration (~1.8 g/L), which was statistically superior to all the other treatments (the top-performing condition across all treatments). This yield significantly exceeded that of other light types under the same photoperiod (full spectrum and cool white), as well as any combination under the 12:12 and 24:0 photoperiods. In contrast, the lowest biomass value (~0.7 g/L) was recorded under cool white light with a 12:12 photoperiod, indicating that this combination was the least favorable for growth. The intermediate performance group was observed mainly under the 24:0 photoperiod, where treatments with red–blue and full spectrum light presented moderate values. Within the same light type, the biomass generally increased from 12:12 to 18:6, with red–blue light showing a marked increase overall under all circumstances. Conversely, the 24 h light combination showed modest improvement over the 12:12 combinations but never reached the level achieved with an 18:6 cycle. This pattern implies that an 18:6 photoperiod, especially in the presence of red–blue light, is closer to ideal for photosynthetic efficiency and cell recovery, whereas a 12:12 photoperiod with white light is the least favorable scenario.

With respect to carbohydrate content (Figure 1b), a distinct pattern from that of biomass was observed. The maximum value was observed under red–blue 12:12, positioning this condition as the most effective for carbohydrate accumulation. Conversely, the lowest value was recorded under full spectrum light with a 24:0 photoperiod, making it the least favorable for this variable. Intermediate results were observed for red–blue 18:6 and cool white 12:12. When grouped by photoperiod, both the 12:12 and 18:6 light ratios favored carbohydrate accumulation, whereas continuous light exposure (24:0) led to a generalized decrease across all light sources. Within each photoperiod, red–blue reached the top group under 12:12, remained intermediate at 18:6, and values for all spectra decreased at 24:0.

In terms of protein content (Figure 1c), the greatest accumulation was again recorded under red–blue light with an 18:6 h photoperiod, with values close to 50% w/w, establishing this configuration as the most favorable for this variable. red–blue 12:12 also showed high values. Cool white 12:12 was intermediate, full spectrum 12:12 was lower, and full spectrum 24:0 showed the minimum. Overall, the three light types under the 18:6 photoperiod consistently outperformed their counterparts under 24:0. Thus, red–blue is not the poorest at 12:12; instead, it belongs among the upper responses.

Finally, regarding lipid accumulation (Figure 1d), a pattern opposite to that of carbohydrates was observed. The highest lipid values (42% w/w) were recorded under red–blue light with both 18:6 and 24:0 photoperiods, and under cool white 24:0, identifying these as the most efficient conditions for lipid production. Intermediate yields were observed under cool white 18:6 and full spectrum 18:6, and the lowest lipid level occurred under full spectrum 12:12. In comparative terms, a clear trend emerges: lipid accumulation increases with extended light exposure, especially under red–blue, so the combination of red–blue light with medium or long photoperiods is both effective and consistent.

The physiological and biochemical responses of Scenedesmus sp. UFPS021 to variations in LED spectral light quality and photoperiod exhibited statistically significant differences across all analyzed variables (Figure 2), enabling the identification of optimal, intermediate, and least favorable conditions.

In terms of biomass concentration (Figure 2a), the highest yield (1.7 g/L) was observed under red–blue light (4:1) with a 24:0 h photoperiod (the top-performing condition). In contrast to Chlorella sp. (Figure 1a), the peak biomass was obtained with this light type but with an 18:6 h photoperiod. In Scenedesmus sp., the peak occurred at red–blue 24:0, red–blue 18:6 ranked next, followed by full spectrum 18:6. Conversely, the lowest values were recorded for cool white 12:12 (~1.0 g/L) and red–blue 12:12, indicating that a balanced photoperiod (12:12), particularly when paired with limited spectral compositions, is the least efficient for promoting cellular growth. Within each light type, a clear trend emerged; biomass increased with longer photoperiods, with 24:0 outperforming 18:6, and both markedly superior to 12:12, especially under red–blue light.

In the case of total carbohydrate content (Figure 2b), the highest value was obtained with red–blue 12:12 (the most favorable configuration for carbohydrate accumulation). In contrast, the lowest carbohydrate content was observed under full spectrum 24:0, with a value close to ~24–25% w/w. Cool white 12:12 belonged to an intermediate range, and the remaining treatments presented moderate and relatively similar levels, forming an intermediate set without highly significant differences. At the photoperiod level, the trend observed in Chlorella sp. was maintained; carbohydrate levels tended to be relatively high under intermittent light (12:12 or 18:6) and decreased under continuous illumination, particularly with full spectrum light. These findings suggest that carbohydrate synthesis in Scenedesmus sp. is more sensitive to photoperiod duration than to spectral quality, with the clear exception being that red–blue 12:12 reached the top response.

In terms of protein content (Figure 2c), the most effective treatments were red–blue 18:6 and red–blue 12:12, both of which reached values of up to ~53% w/w (confirming the strong impact of red–blue light on the stimulation of nitrogen metabolism). Next in rank was red–blue 24:0; cool white 24:0 was intermediate, and cool white 18:6 also occupied an intermediate position. Full spectrum 12:12, full spectrum 18:6, and full spectrum 24:0 formed the lower responses. When grouped by photoperiod, both 12:12 and 18:6 under red–blue lighting were equally effective, suggesting that this strain can achieve maximum protein yield under varying exposure durations, provided that the spectral composition is adequate, whereas full spectrum treatments consistently yielded lower values.

With respect to total lipid accumulation (Figure 2d), the highest values were recorded under red–blue 24:0 and red–blue 18:6, and cool white 24:0 also reached relatively high levels (though below red–blue). Cool white 12:12 and full spectrum 24:0 indicated intermediate performance. Cool white 18:6 and full spectrum 18:6 were lower, and the lowest lipid level occurred under full spectrum 12:12, with red–blue 12:12 falling in the lower–intermediate range. Altogether, lipid accumulation increased with extended light exposure, particularly under red–blue lighting.

4. Discussion

The evaluation timeline in relation to growth phases expressed in this study was aligned with the typical metabolic sequence of photoautotrophic batch cultures. During weeks 1–3, cell expansion and the accumulation of proteins and carbohydrates predominate under intermittent photoperiods, with light-to-biomass conversion rates surpassing those of continuous illumination [10,19]. Under strictly photoautotrophic regimes, nocturnal consumption can reduce early increments of triacylglycerols; therefore, consolidating the carbohydrate and protein balance around day 20 under 12:12 or 18:6 photoperiods is particularly informative [6]. When the available nitrogen in the media decreases and the cell culture enters a slow-growth phase (typically between weeks 3–6) the lipogenesis intensifies, which in turn maximizes the synthesis of lipids after week 4. However, this behavior heavily depends on strain and light conditions [11,12]. This temporal pattern supports the usage of different timeframes for carbohydrates and proteins at day 20 of culture and extend the evaluation of total lipids up to 40 days of culture [10,19]. This behavior agrees with prior optimization of C/N/P (carbon/nitrogen/phosphorus) ratios in the studied strains, which established non-limiting nutrient conditions upon which light modulation was subsequently applied [20].

The attribution of the differential effect on illumination is substantiated by the strict control of other culture variables. Light intensity per unit surface area, working volume, inoculum density, hydrodynamics and mixing, airflow with CO₂ supplementation, and the composition of bold basal medium were all maintained as constants. Experiments were conducted in triplicate, and a two-way ANOVA was applied to disentangle photoperiod and spectrum effects. This approach—systematically varying the light regime while keeping all other factors unchanged—represents the standard methodology for assessing LEDs in microalgae and enables the valid attribution of changes in growth and composition to the light stimulus [21,22,23]. Continuity with the previous C/N/P optimization study on Chlorella sp. UFPS019 and Scenedesmus sp. UFPS021 confirms that, once nutrition is fixed within non-limiting ranges, illumination is the factor directing carbon partitioning toward carbohydrates, proteins, or lipids [20].

For biomass in Chlorella sp. UFPS019, the highest biomass concentration was obtained under red–blue 4:1 with an 18:6 photoperiod (group A, Figure 1a), with the lowest yield under cool white 12:12, around 0.7 g/L (group G). In Scenedesmus sp. UFPS021, the best response was also associated with red–blue light, though under a 24:0 photoperiod (group A, Figure 2a); this was followed by red–blue 18:6 (group B) and full spectrum 18:6 (group BC), with minima observed under cool white 12:12 (group F) and red–blue 12:12 (group DE). This discrepancy in optimal photoperiods between Chlorella sp. (18:6) and Scenedesmus sp. (24:0) is consistent with intergeneric differences in tolerance to prolonged exposure and in photochemical repair kinetics. Morphotypes of Scenedesmus sp. have been reported to sustain higher growth rates under extended photoperiods with dominant red components when all other conditions remain constant [11,22]. In Chlorella sp., multiple controlled comparisons rank red–blue mixtures above white light for biomass productivity when peaks of chlorophyll a and b absorption (near 450 and 660 nm) are synchronized and when a sufficient dark phase is preserved for recovery [12,24]. In both cases, 24:0 did not surpass 18:6 in Chlorella sp., consistent with reports documenting no net gains under continuous light due to insufficient nocturnal recovery [25], and with evidence of higher specific costs per unit product at elevated daily light doses in comparable configurations [26].

In Chlorella sp., carbohydrate content reached its maximum under red–blue 12:12 (group A, Figure 1b) and its minimum under full spectrum 24:0 at ~25% w/w (group G). In Scenedesmus sp., the pattern was analogous: a maximum under red–blue 12:12 (group A, Figure 2b) and a minimum under full spectrum 24:0 at 24-25% w/w (group E), with cool white 12:12 in groups B–C and the remaining treatments forming intermediate sets. The appearance of maxima under 12:12 aligns with the role of the dark phase in channeling carbon toward starch and other storage polysaccharides, as demonstrated in Chlorella sp. under intermittent regimes [6], and with models showing reduced net carbohydrate accumulation under continuous illumination due to the increased respiration and mobilization of reserves [19]. The persistence of intermediate-to-high values under red–blue 18:6 in Chlorella sp. is consistent with responses of strains adapted to high irradiance that sustain elevated carbohydrates under nitrogen limitation when the photoperiod is extended to medium intervals [27]. Meanwhile, in Scenedesmus sp., the superiority of red–blue versus white or full spectrum for carbohydrate accumulation under intermittent cycles has been reported in comparable designs [22,23].

The highest protein content in Chlorella sp. (~50% w/w) was recorded under red–blue 18:6 (group A, Figure 1c); red–blue 12:12 remained in the upper AB group, cool white 12:12 was placed in CD, full spectrum 12:12 in EF, and full spectrum 24:0 showed the lowest value in F. In Scenedesmus sp., the most effective treatments were red–blue 18:6 and 12:12, both in group A with ~53% w/w; red–blue 24:0 was in group B; cool white 24:0 in group C; and cool white 18:6 in BC; full spectrum 12:12, 18:6, and 24:0 fell into groups D–E. The superiority of red–blue mixtures over white spectra for protein fraction aligns with experiments in which blue light enhances nitrogen assimilation and protein synthesis while red light sustains overall productivity [9]. Furthermore, under wastewater treatment conditions, Chlorella sp. increases protein and pigment levels with blue components at controlled intensities [25]. The convergence of both strains around red–blue with 12:12–18:6 photoperiods for protein reinforces that intermittent cycles favor nocturnal reprogramming of the photosynthetic apparatus and nitrogen reassignment toward structural and enzymatic proteins [10].

The highest lipid values in Chlorella sp. (~42% w/w) were observed under red–blue 18:6 and 24:0, and also under cool white 24:0, all grouped in A (Figure 1d). Intermediate responses corresponded to cool white 18:6 and full spectrum 18:6 (groups B–C), while the minimum occurred under full spectrum 12:12 (group D). In Scenedesmus sp., maxima were recorded under red–blue 24:0 and 18:6, both in group A (Figure 2d), with cool white 24:0 also in A; intermediate levels were observed under cool white 12:12 and full spectrum 24:0 (groups B and C); low responses under cool white 18:6 and full spectrum 18:6 (groups C and D); and the lowest under full spectrum 12:12 (group F), with red–blue 12:12 in CE. The preference of both strains for red–blue mixtures with medium-to-prolonged photoperiods for triacylglycerol accumulation coincides with reports that red light sustains the supply of reducing power and acyl-CoA precursors, while blue light co-activates lipogenic genes [12,28]. At pilot scales, high lipid fractions have been achieved with intermediate photoperiods at moderate intensities without requiring maximum biomass, as in Verrucodesmus verrucosus under 12:12 and ~2000 lux with 50.4% lipids [29], supporting the sensitivity of lipogenesis to light’s temporal architecture rather than to total dose alone. Results in saline–alkaline matrices also show that red light increases lipids and that color combinations outperform monochromatic conditions [30], consistent with the patterns observed here.

Differences between strains and light conditions are also explained by interactions among spectrum, photoperiod, and internal light transfer. Attenuation across culture depth, self-shading, and irradiance gradients depend on the geometry and mixing regime; configurations that reduce gradients or generate benign pulsed exposure increase the usable fraction of light and limit photoinhibition [31]. Temporal modulation of light delivery—frequency and duty cycle—can optimize the coupling between closure–opening times of reaction centers and photon supply, yielding productivity gains per photon [32].

The observation that Chlorella sp. maximizes biomass under red–blue 18:6 while Scenedesmus sp. does so under red–blue 24:0, despite identical nutrition, hydrodynamics, and inoculum density, confirms a primary effect of light on strain-specific carbon partitioning pathways. This interpretation aligns with controlled comparisons where medium and operation are kept constant, attributing changes in growth and composition to light quality and regime [21,22], and with the precedent of C/N/P optimization in these same strains, which avoided nutritional limitations and provides continuity to the present exploration of photoperiods and spectra [20]. Thus, the observed differences are explained by photo acclimatory requirements and distinct tolerances to prolonged exposure rather than by nutritional or operational confounders.

From an applied perspective, the spectrum–photoperiod windows defined here allow structuring production trains oriented toward target metabolites and comparable with platforms already implemented in biorefinery. For carbohydrate- and protein-rich streams, Chlorella sp. under red–blue 18:6 and Scenedesmus sp. under red–blue 12:12 enable harvests at 20 days with fractions of 27–48% w/w carbohydrates and 50–53% w/w proteins, suitable for fermentable sugars and functional flours, consistent with starch accumulation and superior biomass/light balances under light–dark cycles [6,10,19]. For oils, Chlorella sp. under red–blue 18:6 and 24:0 and Scenedesmus sp. under red–blue 24:0 reach ~42% w/w lipids, an operational range within values exploited in biodiesel schemes using oleaginous microalgae and close to reference records in pilot systems such as V. verrucosus with 50.4% under 12:12 at moderate intensities [29,32]. Moreover, comparative studies show that red–blue mixtures outperform monochromatic conditions in promoting TAG, and that the blue component co-activates lipogenic pathways, reinforcing the use of these regimes for lipid “finishing” stages [28,30]. In process integration, the results enable two pragmatic configurations: a biofuel-oriented line with a growth phase under red–blue 18:6 followed by a 24:0 pulse to maximize TAG before day 40; a coproduct line for food and platform derivatives with 20-day harvests under red–blue 12:12–18:6 for carbohydrate- and protein-rich concentrates, coupled to CO₂ capture and effluent valorization, consistent with biorefinery evaluations in Chlorella sp. recommending coproduct integration to improve technical and environmental indicators [20,32]. This programmability of spectrum and photoperiod is transferable to commercial LED photobioreactors through cycle control and spectral switching, enabling metabolite-specific production campaigns supported by verified accumulation kinetics and photon productivity benchmarks under light–dark regimes [10,32].

5. Conclusions

The analysis of the physiological and biochemical responses of both strains confirmed that the yield of each metabolite (and, consequently, the overall efficiency) did not depend on a single factor: only light quality or photoperiod duration. The configurations that use red–blue light with intermediate photoperiods (18:6 h) appear to be the most balanced between high photosynthesis and metabolic regeneration in the dark phase, optimizing productivity relative to the consumption of energy. On the other hand, continuous light (24:0 h) significantly increased the levels of some metabolites, especially lipids, but with high energy waste during the intensive process and a decrease in important valuable compounds, which makes operability not feasible for large-scale production. It is a reminder not only to consider endpoint concentration values in the results but also the product of these values by energy input.

For Chlorella sp., the mixed light increased the carbohydrate content under SS conditions, whereas the red and blue lights resulted in greater protein production, maintaining the best use efficiency for the PS of available energy. Moreover, with respect to the photoperiod regime, red–blue light significantly increased the protein content in Scenedesmus sp. under both different photoperiod regimes and continuous illumination; however, this increased energy consumption led to high lipid accumulation. The results confirm that optimal conditions are not determined only by the maximum of a single metabolite but also by its integration into a bioprocess model that promotes physiological stability, resource-efficient use, and energetic balance, enabling sustainable production with broad applicability in future biorefinery scenarios.