Trade-Off Between Growth Regimes in Chlorella vulgaris: Impact on Carotenoid Production

Abstract

With the increasing awareness of socio-environmental issues, a global trend has emerged emphasizing the valorization of natural ingredients that promote health and well-being within sustainable production systems, such as microalgae-based carotenoids. Currently, little is understood about the correlation between biomass productivity and carotenoid content, which is a fundamental parameter for facilitating the immediate expansion of microalgae bioprocesses and ensuring the availability and industrial viability of these compounds. In this context, this study aims to investigate the carotenoid profile of Chlorella vulgaris through growth curve experiments conducted under photoautotrophic and heterotrophic regimes. Additionally, a trade-off analysis was performed for the production of carotenoids from microalgae. Carotenoids were quantified using high-performance liquid chromatography coupled with diode array and mass spectrometry detectors (HPLC-PDA-MS/MS). The performance of kinetic phases and energy demands across growth regimes was assessed to provide insights into production trade-offs. The results indicated that a total of 22 different carotenoids were identified in all the extracts. The all-trans-lutein and all-trans-β-carotene were the majority compounds. The total carotenoid content of Chlorella vulgaris revealed significant differences in the kinetic phases of carotenoid production, indicating that carotenoid volumetric production is only viable if the cultures are conducted until the log and stationary phases, based on the function of the biomass volumetric production (weight.volume⁻¹). Therefore, the best trade-off for the process was to provide photoautotrophic growth until the exponential phase (log). Under this condition, the maximum carotenoid and lutein content was 2921.70 µg.L⁻¹, reaching a maximum cell biomass of 1.46 g.L⁻¹. From an environmental/economic point of view, the energy demand was 7.74 kWh.L⁻¹. Finally, the scientific advances achieved in this study provide a holistic view of the influence of the main cultivation methods on the production of microalgae carotenoids, suggesting a viable initial direction for different industrial applications.

1. Introduction

Along with the growing global awareness of socio-environmental issues within the industrial sector, significant changes have also occurred in the preferences of the population [1,2]. These transformations are linked to a continuous increase in the popularity of natural ingredients that promote health, well-being, and quality of life within sustainable production systems. Consequently, the exploration of bioactive and functional biomolecules, such as natural carotenoids, paves the way for a new era of sustainable bioingredients and bioproducts [3,4]. These molecules play a notable role as antioxidants and precursors of vitamin A and in eye health. In addition, research has suggested their role as prebiotics, reducing cardiovascular diseases, obesity, diabetes, and cancer [5].

From this perspective, microalgae biotechnology assertively positions itself in the current global scenario, aligning with the principles of One Health. This is a consequence of the metabolic versatility of algae biomass, which allows for a wide range of applications in the industrial sector, including human and animal nutrition, cosmetics, and medicine [4,6]. In addition, with biotechnology enhancement, the industrial production of fine chemical compounds has boosted the microalgae market. Estimates indicate that approximately 35.82 million tons per year of microalgae biomass is produced globally, totaling a value of 11.8 billion BRL. Modeling projects a market valued at 25.4 billion BRL by 2033 [7]. However, from these total numbers, it is estimated that 75% is directed toward the production of microalgae pigments, including carotenoids such as β-carotene, astaxanthin, and lutein [8].

Nevertheless, although carotenoids have reached consumers, the current biotechnological routes do not meet market demands. This occurs mainly due to the volumetric productivity of cultures. Also, another key aspect that directly influences this bottleneck is the understanding of the growth regimes and phases for the production of compounds of interest [9]. Therefore, investigating the carotenoid biosynthesis under the main growth regimes—autotrophic and heterotrophic—will help define substrate bioconversion patterns and direct the regimes and cultivation phases according to the intended interest.

Currently, little is known about the correlation between biomass productivity and carotenoid content, which is a fundamental parameter to facilitate the immediate expansion of microalgae bioprocesses and ensure the availability and industrial viability of these biocompounds. Most of the work available in the literature focuses only on specific carotenoids, disregarding the profile of carotenoids in their entirety. The reports available are limited to stoichiometric restrictions of cultures [10], different substrate sources [11], and surveys associated with bioaccessibilities and cellular uptakes [12,13].

In addition, although studies on carotenoid content under photoautotrophic and heterotrophic growth regimes with Chlorella vulgaris have been reported in the literature, most of these present only quantitative values of the total carotenoid profile or information from a photodiode array, without addressing characterization by mass spectrometry (MS) in detail [14,15]. In the carotenoid analysis, mass spectrometry is focused on identification, while other methods do not allow for the distinction between structures with the same UV–visible spectra and similar chromatographic behavior, or when the acquisition of high-quality UV–visible spectra is not possible in low-concentration samples. For this reason, the application of MS techniques to the analysis of carotenoids in phytoplankton has expanded the knowledge on the composition and metabolism of carotenoids [16].

These contributions are relevant to the literature and serve as building blocks for advancing to a new level in microalgae biotechnology. However, these previous approaches analyze one analytical stage or a dyad; when observing the evolutionary context of research, development, innovation, and industry, it is understood that this approach brought insufficient results to, on the whole, consolidate the future demands for microalgal-based pigment production.

Against this background, the rationale behind this study and the main objective of this research is to analyze, using HPLC-PDA-MS/MS, the qualitative and quantitative profile of carotenoids from Chlorella vulgaris under comparative growth regimes, photoautotrophic and heterotrophic, correlating with the growth phases and volumetric productivities. Also, to establish the industrial trade-off in the production of microalgae-based carotenoids, biosynthetic challenges including the promotion of viable pathways for the industrial consolidation of microalgae biotechnology, industrial viability, and market opportunities were discussed. Therefore, the discoveries found in this work will serve as the cornerstone to help overcome the current limitations and provide important reference data for studies on metabolic pathway effects, growth screening, and culture selection, opening pathways for full-fledged industrial exploration of microalgae carotenoids.

2. Material and Methods

2.1. Chemicals

The standards of all-trans-lutein, all-trans-β-cryptoxanthin, and all-trans-β-carotene (purity ≥ 98%, HPLC) were purchased from Sigma-Aldrich (Darmstadt, Germany). Methanol (MeOH) and methyl tert-butyl ether (MTBE) (both of HPLC-grade) and the analytical-grade solvents, ethanol, acetone, ethyl acetate, petroleum ether, and diethyl ether, were purchased from Merck (Darmstadt, Germany).

2.2. Microalgae Culture and Biomass Production

Axenic cultures of Chlorella vulgaris (CPCC90) were used in the experiments. Stock cultures were propagated and maintained in synthetic BG-11 medium (Braun–Grunow medium) [17]. The incubation conditions were a temperature of 25 °C, a photon flux density of 30 μmol.m⁻².s⁻¹, and a photoperiod of 12/12 h light/dark.

The biomass productions were made under photoautotrophic and heterotrophic growth regimes. Photoautotrophic growth regimes were performed in a bubble column photobioreactor [18] operating under a batch regime, fed on 2.0 L of BG-11 medium. The experimental conditions were as follows: an initial cell concentration of 100 mg.L⁻¹, an isothermal reactor operating at a temperature of 25 °C, a photon flux density of 150 μmol.m⁻².s⁻¹, continuous aeration of 1 VVM (volume of air per volume of culture per minute), and continuous lighting conditions of 24:0 h light/dark.

For the heterotrophic growth regimes, the experiments were developed in a bioreactor operating in the batch system, fed with 2.0 L of culture medium. The experimental conditions were as follows: an initial inoculum concentration of 100 mg.L⁻¹, an isothermal reactor operating at a temperature of 25 °C, a pH adjusted to 7.6, aeration of 1 VVM, and the absence of light. The culture medium consisted of BG11 supplemented with an exogenous carbon source to obtain a fixed C/N ratio of 20. The monosaccharide concentration was adjusted stoichiometrically, using D-glucose (12.4 g.L⁻¹). The glucose was weighed and diluted in synthetic BG-11 medium, followed by autoclaving at 121 °C for 20 min [19].

In both growth regimes, the biomasses were separated from the culture medium by centrifugation (1500× g; 10 min; and 10 °C), the supernatants were discarded, and the wet biomasses (95% moisture) were stored immediately in closed containers protected from light, under refrigeration.

2.3. Experimental Setup and Kinetic Parameters

The monitoring of the kinetic growth phases was performed every 24 h. Beginning at the control zero time, there was a 24 h lag phase, 48–72–96 h log phase, and 120 h stationary phase for photoautotrophic cultivation. In the heterotrophic culture, the measurements were conducted in the periods at zero time, 04–08–16–20 and 24 h for the lag phase, 48–72–96 h for the log phase, and 120 h for the stationary phase. All the experiments were performed in duplicate; therefore, the kinetic data refer to the mean value of the replicates.

The biomass data were used to calculate the biomass productivity (g.L.d⁻¹), the maximum specific growth rate (d⁻¹), and the generation time (d) from Equations (1)–(3), respectively:

$$\mathrm{Px}={\displaystyle \frac{(X_i-{\mathrm{X}}_{\mathrm{i}-1})}{(t_i-{\mathrm{t}}_{\mathrm{i}-1})}}$$

(1)

$$ln\left({\displaystyle \frac{Xi}{X0}}\right)=\mu max\times t$$

(2)

$$Tg={\displaystyle \frac{0.693}{\mu max}}$$

(3)

where X_i is the biomass concentration at time t_i (g.L⁻¹) and X_i−1 is the biomass concentration at time t_i−1 (g.L⁻¹), and t is the residence time (d). The residence time was defined as the elapsed time to reach maximum cell biomass.

2.4. Measurement of Biomass

The cell biomass was determined gravimetrically by filtering a known culture volume through a 0.45 μm membrane filter (Millex FG^®, Billerica, MA, USA), and then drying was conducted at 50 °C until constant weight (24 h). At each weighing, the filter containing the biomass was kept in a desiccator and subsequently weighed on an analytical balance.

2.5. Carotenoids Profile

The carotenoids were exhaustively extracted according to Rodrigues et al. (2015). They were analyzed by HPLC (Shimadzu, Kyoto, Japan) using a diode array detector (PDA) (model SPD-M20A) and a mass spectrometer with an ion-trap analyzer and atmospheric pressure chemical ionization (APCI) source (model Esquire 4000, Bruker Daltonics, Bremem, Germany). The carotenoid separation was performed on a C30 YMC column (5 μm, 250 × 4.6 mm) (Waters, Wilmington-DE, USA). Prior to the HPLC-PDA analysis, the carotenoid extract was solubilized in methanol (MeOH): methyl tert-butyl ether (MTBE) (70:30) and filtered through Millipore membranes (0.22 μm). The mobile phases were A (MeOH) and B (MTBE), using a linear gradient program as follows: from 0 to 30 min 5% B; from 30 to 40 min, 5 to 30% B; from 40 to 41 min, 30 to 50% B; and from 41 to 50 min, 50 to 5% B. The flow rate was set at 0.9 mL.min⁻¹, the injection volume was 20 μL, the column temperature was maintained at 22 °C, the UV/vis spectra were acquired between 300 and 700 nm, and the chromatograms were processed at 450 nm.

The MS/MS analysis was achieved according to [20] with adaptations, and the APCI interface operated in positive (+) mode with the following: detector voltage: 4.5 kV; interface temperature: 350 °C; DL temperature: 250 °C; heat block temperature: 200 °C; nebulizing gas flow (N₂): 3.0 L.min⁻¹; drying gas flow (N₂): 5.0 L.min⁻¹; collision-induced dissociation (CID) gas: 23 kPa (argon); and event time: 0.5 s. To improve the quality of identification, MS/MS was used simultaneously in the SIM (Select Ion Monitoring) and MRM (Multiple Reaction Monitoring) modes. The identification was performed according to the following combined information: elution order on a C30 HPLC column, co-chromatography with authentic standards, UV–visible spectrum, and mass spectral characteristics, which were compared with the data available in the literature. The carotenoids were also quantified by HPLC-PDA, using five-point analytical curves (all-trans-violaxanthin, all-trans-zeaxanthin, all-trans-lutein, all-trans-α-carotene, and all-trans-β-carotene).

2.6. Evaluation of Energy Requirements for an Industrial Trade-Off in Microalgal-Based Pigments Production

The energy demand of the microalgal process was established as the energy necessary for the full functioning of the cultivation systems during the hydraulic residence time and, subsequently, obtaining the final pigment. For the photoautotrophic regime, the energy required for aeration, carbon dioxide injection, and lighting of the photobioreactor was encompassed, as described by [18]. On the other hand, for the heterotrophic regime, agitation and sterilization of the bioreactor were considered [21]. Furthermore, for both regimes, the energy requirements for downstream steps, such as centrifugation, drying, and extraction of compounds, were considered, as described by [22]. The functional unit was considered kWh.L⁻¹.

2.7. Statistical Analysis

The analysis was performed using Statistica 7.0 software (Statsoft, Tulsa, OK, USA). The significance of the experimental data for the kinetic parameters was determined using Student’s t-test (p < 0.05). For the remaining data, the significance was assessed through one-way ANOVA followed by Tukey’s test (p < 0.05). The normality of the data was previously evaluated using the Shapiro–Wilk test.

3. Results

3.1. Identification of the Carotenoid Profile in All Growth Regimes

The chromatographic and spectrometric characteristics of the different carotenoids separated during phototrophic and heterotrophic growth are shown in

Table 1. Chromatographic, UV/Vis, and mass spectrometry characteristics of Chlorella vulgaris carotenoids, obtained by HPLC-PDA-MS/MS.

Peak a	Pigments	tR (min) b	UV-Vis Characteristics				Fragment Ions (Positive Mode) (m/z)
			λmáx (nm) c	III/II d (%)	AB/II e (%)	[M + H]+	MS/MS
1	15-cis-neochrome	6.2	327, 405, 428, 455	65	42	601	583 [M + H − 18]+, 565 [M + H − 18 − 18]+, 547 [M + H − 18 − 18 − 18]+, 509 [M + H − 92]+
2	13-cis-neoxanthin	6.7	337, 419, 443,471	81	17	601	583 [M + H − 18f, 547 [M + H − 18 − 18 − 18]+, 221
3	all-trans-neoxanthin	7.5	415, 438, 468	78	0	601	583 [M + H − 18]+, 565 [M + H − 18 − 18]+, 547 [M + H − 18 − 18 − 18]+, 509 [M + H − 92]+
4	all-trans-neochrome	7.7	398,421, 447	80	0	601	583 [M + H − 18]+,, 547 [M + H − 18 − 18 − 18]+, 221
5	9-cis- neoxanthin	8.0	325, 415, 438, 467	78	0	601	583 [M + H − 18]+, 565 [M + H − 18 − 18]+, 509 [M + H − 92]+
6	13-cis-antheraxanthin	8.7	326, 415, 438, 467	72	13	585	583 [M + H − 18]+, 565 [M + H − 18 − 18]+, 509 [M + H − 92]+
7	all-trans-luteoxanthin	8.8	400, 420, 447	100	0	601	583 [M + H − 18]+
8	all-trans-antheraxanthin	9.3	416, 442, 473	50	0	585	567 [M + H − 18]+, 549 [M + H − 18 − 18]+, 531
9	15-cis-lutein	10.3	331, 415, 437, 465	37	31	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+
10	13-cis-lutein	11.2	328, 415, 438, 465	25	39	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+, 477 [M + H − 92]+
11	all-trans-lutein	12.3	419, 443, 471	57	0	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+
12	all-trans-zeaxanthin	14.5	425, 449, 475	25	0	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+,495, 477 [M + H − 92]+, 459
13	9-cis-lutein	15.2	326, 420, 440, 465	50	12	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+,495, 477 [M + H − 92]+, 459
14	9-cis-zeaxanthin	17.6	338, 420, 445, 470	33	25	569	551 [M + H − 18]+, 533 [M + H − 18 − 18]+,495, 477 [M + H − 92]+, 459
15	2′-dehydrodeoxymyxol	20.2	445, 473, 504	63	0	567	549 [M + H − 18]+
16	5,6-epoxy-β-carotene	20.3	420, 446, 470	50	0	553	535 [M + H − 18]+, 461 [M + H − 92]+, 205
17	all-trans-echinenone	23.6	462	nc f	0	551	533 [M + H − 18]+, 427, 203
18	9-cis-echinenone	25.6	342, 450	nc	20	551	533 [M + H − 18]+, 427, 203
19	all-trans-α-carotene	27.8	420, 445, 473	62	0	537	444 [M + H − 92]+, 399, 355
20	9-cis- α-carotene	28.9	330, 420, 444, 472	70	nc	537	444 [M + H − 92]+, 399, 355
21	all-trans-β-carotene	31.0	425, 451, 476	25	0	537	444 [M + H − 92]+, 399, 355
22	9-cis-β-carotene	33.7	341, 420, 446, 472	20	14	537	444 [M + H − 92]+, 399, 355

^a Numbered accordingly. ^b t_R: Retention time on the C30 column. ^c Linear gradient MeOH:MTBE. ^d Spectral fine structure: Ratio of the height of the longest wavelength absorption peak (III) and that of the middle absorption peak (II). ^e Ratio of the cis peak (A_B) and the middle absorption peak (II). ^f Not calculated.

. The carotenoids were identified based on the combined information obtained from chromatographic elution, co-chromatography with standards, and UV–visible and mass spectra characteristics. A description of microalgae carotenoid identification using chromatographic information from HPLC-PDA-MS/MS (APCI positive mode) was previously described in detail in the literature [4,13,23,24,25,26,27].

In general, a total of twenty-two carotenoids were separated under the photoautotrophic or heterotrophic growth conditions, of which most were all-trans-α-carotene- or all-trans-β-carotene-derived structures, except 2′-dehydrodeoxymyxol, synthesized through cyclization, ketolation, cis-isomerization, hydroxylation, acetylation, epoxidation, and epoxide-furanoxide rearrangement reactions (Figure 1).

3.2. Determination of the Carotenoid Profile During Phototrophic Regime Growth

Table 2. Quantitative characterization of carotenoids in kinetic growth phases (μg.g⁻¹ dry weight).

Peak	Compounds	Phototrophic *				Heterotrophic *
		Zero-Time	Lag	Log	Stacionary	Zero-Time	Lag	Log	Stacionary
1	15-cis-neochrome	nd 1	nd	nd	nd	nd	36.31 ± 0.03 a	5.33 ± 0.02 b	nd
2	13-cis-neoxanthin	nd	nd	nd	nd	nd	22.41 ± 0.04 a	nd	nd
3	all-trans-neoxanthin	119.80 ± 0.82 b	170.89 ± 0.49 a	98.47 ± 0.10 c	99.07 ± 0.27 c	16.72 ± 0.07 g	61.50 ± 0.16 d	20.45 ± 0.02 f	28.31 ± 0.53 e
4	all-trans-neochrome	nd	nd	nd	nd	nd	nd	5.00 ± 0.00 a	nd
5	9-cis-neoxanthin	65.17 ± 0.09 d	103.11 ± 0.13 a	84.47 ± 0.07 c	98.47 ± 0.48 b	28.89 ± 0.00 f	52.74 ± 0.18 e	18.23 ± 0.15 h	22.50 ± 0.02 g
6	13-cis-antheraxanthin	72.44 ± 0.19 b	85.15 ± 0.21 a	nd	nd	nd	21.35 ± 0.13 c	12.69 ± 0.13 e	19.55 ± 0.17 d
7	all-trans-luteoxanthin	nd	nd	27.90 ± 0.46 b	81.60 ± 0.19 a	24.34 ± 0.09 c	24.28 ± 0.08 c	nd	nd
8	all-trans-antheraxanthin	nd	nd	nd	nd	nd	40.73 ± 0.18 a	5.35 ± 0.00 c	16.91 ± 0.01 b
9	15-cis-lutein	54.64 ± 0.14 b	44.79 ± 0.79 c	46.89 ± 0.60 c	59.31 ± 0.31 a	45.28 ± 0.08 c	30.42 ± 0.42 d	5.01 ± 0.13 f	11.51 ± 0.02 e
10	13-cis-lutein	27.08 ± 0.44 a	20.55 ± 0.31 b	15.03 ± 1.09 d	18.37 ± 0.13 c	21.78 ± 0.07 b	11.44 ± 0.15 e	3.27 ± 0.13 f	4.62 ± 0.12 f
11	all-trans-lutein	1054.15 ± 0.98 a	510.83 ± 1.25 d	654.05 ± 3.77 c	700.53 ± 1.81 b	96.68 ± 0.39 h	330.53 ± 2.94 f	236.46 ± 1.33 g	363.02 ± 0.69 e
12	all-trans-zeaxanthin	61.28 ± 0.10 d	85.88 ± 0.06 c	167.62 ± 1.17 a	161.04 ± 0.82 b	18.61 ± 0.02 f	50.53 ± 0.20 e	5.55 ± 0.01 g	nd
13	9-cis-lutein	14.53 ± 0.09 b	7.34 ± 0.19 d	8.26 ± 0.23 c,d	10.62 ± 0.31 c	64.19 ± 0.03 a	13.63 ± 0.24 b	6.89 ± 0.11 d	15.26 ± 0.17 b
14	9-cis-zeaxanthin	nd	nd	nd	nd	53.01 ± 0.60 a	41.62 ± 0.28 b	11.87 ± 0.07 d	18.32 ± 0.05 c
15	2′-dehydrodeoxymyxol	nd	nd	nd	nd	nd	16.21 ± 0.56 a	nd	nd
16	5,6-epoxy-β-carotene	nd	nd	nd	nd	nd	18.05 ± 0.35 a	nd	nd
17	all-trans-echinenone	nd	114.53 ± 0.36 c	266.67 ± 2.01 a	232.16 ± 2.41 b	nd	49.69 ± 0.16 e	nd	nd
18	9-cis-echinenone	nd	112.78 ± 0.52c	159.79 ± 1.64 a	137.44 ± 2.56 b	nd	45.61 ± 0.98 e	nd	nd
19	all-trans-α-carotene	103.42 ± 0.01 a	5.21 ± 0.20 f	27.87 ± 0.42 d	22.00 ± 0.24 d	72.23 ± 1.48 b	41.43 ± 0.68 c	13.93 ± 0.46 e	24.22 ± 0.18 d
20	9-cis-α-carotene	3.06 ± 0.12 a	nd	nd	nd	6.87 ± 0.40 a	nd	nd	nd
21	all-trans-β-carotene	214.22 ± 0.92 c	192.05 ± 0.49 d	311.64 ± 1.47 a	296.59 ± 1.08 b	92.82 ± 2.56 f	170.43 ± 1.05 e	31.81 ± 0.28 h	48.42 ± 0.02 g
22	9-cis-β-carotene	74.62 ± 0.70 d	87.63 ± 0.08 c	132.51 ± 0.83 a	117.79 ± 0.66 b	30.12 ± 0.43 f	48.81 ± 0.36 e	5.85 ± 0.03 h	17.86 ± 0.34 g
Total		1849.88 ± 0.80 c	1540.74 ± 1.76 d	2001.17 ± 8.50 b	2034.99 ± 0.92 a	571.54 ± 5.07 g	1127.72 ± 7.25 e	387.69 ± 2.33 h	590.51 ± 1.91 f

¹ Not detected. * Values are average and standard deviation of triplicates. Values (rows) followed by different superscript letters indicate statistical differences (p < 0.05).

presents the quantitative profiles of the carotenoids identified under the photoautotrophic growth conditions. Twelve carotenoids were identified at zero time from phototrophic conditions, including three epoxycarotenoids (peaks 3, 5, and 6), five hydroxycarotenoids (peaks 9, 10, 11, 12, and 13), and four carotenes (peaks 19, 20, 21, and 22). All-trans-lutein (1054.15 μg.g⁻¹, peak 11) and all-trans-β-carotene (214.22 μg.g⁻¹, peak 21) were the major ones, representing 68.03% of the total carotenoid content, followed by all-trans-neoxanthin (6.43%) and all-trans-α-carotene (5.55%) as the major carotenoids at zero time.

Among the 12 carotenoids identified at zero time from the phototrophic growth conditions, 11 compounds were common to the lag phase profile (peaks 3, 5, 6, 9, 10, 11, 12, 13, 19, 20, 21, and 22), and two compounds were characterized only after the lag phase (peaks 17 and 18).

Considering the quantitative profile, it was observed that the total amount of carotenoids decreased from zero time (1849.88 μg.g⁻¹) until the microalgae reached the lag phase (1540.74 μg.g⁻¹). This behavior is due to the response of the main carotenoids all-trans-lutein (510.83 μg.g⁻¹) and all-trans-β-carotene (192.05 μg.g⁻¹) to the continuous illumination.

In contrast, the log phase (2001.17 μg.g⁻¹) significantly increased the content of most carotenoids compared to the lag phase (1540.74 μg.g⁻¹) in the following order: all-trans-lutein (654.05 μg.g⁻¹) > all-trans-β-carotene (311.64 μg.g⁻¹) > all-trans-echinenone (266.67 μg.g⁻¹) > all-trans-zeaxanthin (167.62 μg.g⁻¹) > 9-cis-echinenone (159.79 μg.g⁻¹) > 9-cis-β-carotene (132.51 μg.g⁻¹) > all-trans-α-carotene (27.87 μg.g⁻¹). Meanwhile, all-trans-neoxanthin (98.47 μg.g⁻¹), 9-cis-neoxanthin (84.47 μg.g⁻¹), and 13-cis-antheraxanthin (not detected) showed a significant decrease concerning the lag phase. On the other hand, 15-cis-lutein (46.89 μg.g⁻¹) and 9-cis-lutein (8.26 μg.g⁻¹) showed no significant differences compared to the lag phase. Moreover, all-trans-luteoxanthin (27.90 μg.g⁻¹) was not detected at zero time and in the lag phase but was identified in the log phase.

Comparatively to the other growth stages (lag and log), the highest total carotenoid content in the dried biomass was determined in the stationary phase (2034.99 μg.g⁻¹) (p < 0.05).

As for the carotenoids detected in the stationary phase when compared to the lag phase, all-trans-lutein (700.53 μg.g⁻¹), all-trans-β-carotene (296.59 μg.g⁻¹), all-trans-echinenone (232.16 μg.g⁻¹), all-trans-zeaxanthin (161.04 μg.g⁻¹), 9-cis-echinenone (137.44 μg.g⁻¹), 9-cis-β-carotene (117.79 μg.g⁻¹), 15-cis-lutein (59.31 μg.g⁻¹), all-trans-α-carotene (22.00 μg.g⁻¹), and 9-cis-lutein (10.62 μg.g⁻¹) increased their contents in the C. vulgaris culture, even with the stationary growth. Nonetheless, the contents decreased for all-trans-neoxanthin (99.07 μg.g⁻¹), 9-cis-neoxanthin (98.47 μg.g⁻¹), and 13-cis-lutein (18.37 μg.g⁻¹) (compared to the lag phase). Moreover, all-trans-luteoxanthin (81.60 μg.g⁻¹) was not detected in the lag phase but was identified in the stationary phase.

3.3. Determination of the Carotenoid Profile During Heterotrophic Regime Growth

Thirteen carotenoids were identified at zero time from the heterotrophic conditions (see Table 2), presenting a total carotenoid content of 571.54 μg.g⁻¹. As in the photoautotrophic conditions, all-trans-lutein (96.68 µg.g⁻¹) and all-trans-β-carotene (92.82 µg.g⁻¹) were the largest carotenoids, followed by all-trans-α-carotene (72.23 µg.g⁻¹).

In this regime, the lag phase exhibited the most complete profile constituted by twenty carotenoids. The major carotenoids were the same ones detected at zero time, all-trans-lutein (330.53 μg.g⁻¹, peak 11) and all-trans-β-carotene (170.43 μg.g⁻¹, peak 21), which corresponds to 44.42% of the fraction of carotenoids in the extract. Unlike the results described at zero time, the lag phase presented three ketocarotenoids (peaks 15, 17, and 18), eight epoxycarotenoids (peaks 1, 2, 3, 5, 6, 7, 8, and 16), six hydroxycarotenoids (peaks 9, 10, 11, 12, 13, and 14), and three carotenes (peaks 19, 21, and 22).

Of particular interest, the lag phase of the heterotrophic growth promoted the production of unique carotenoids from the microalgae (peaks 15, 17, and 18) that can probably potentiate the antioxidant activity of the extract in the lag phase. In addition, the log phase led to a pronounced reduction in the total carotenoid contents (387.69 μg g⁻¹). Thus, all the carotenoids had a significant quantitative reduction. The concentration of all-trans-β-carotene decreased by 81%. This decrease was extended to a lower degree for all-trans-lutein (28%).

On the other hand, in the stationary phase, most carotenoids had a significant quantitative reduction, except all-trans-lutein and 9-cis-lutein. In addition, about the log phase, most carotenoids showed a higher concentration, except for peaks 1, 4, and 12, which were no longer detected in this growth phase.

3.4. Growth Curve Performance and Industrial Trade-Off in Microalgal-Based Pigment Production

Table 3. Growth kinetics for Chlorella vulgaris under photoautotrophic and heterotrophic growth regimes.

Parameter	Photoautotrophic	Heterotrophic
Xmax (g/L)	1.90 b ± 0.01	2.20 a ± 0.02
µmax (d−1)	0.51 a ± 0.00	0.45 b ± 0.00
Tg (d)	1.35 b ± 0.01	1.54 a ± 0.01
Px (average) (g/L/d)	0.23 b ± 0.02	0.24 a ± 0.02
Px (peak) (g/L/d)	0.36 b ± 0.01	0.42 a ± 0.01

X_max: maximum cell biomass; µ_max: maximum specific growth rate; Tg: cell generation time; and Px: cell productivity.

presents the kinetic growth parameters for Chlorella vulgaris under a photoautotrophic and heterotrophic growth regime.

Under the autotrophic regime, the microalga Chlorella vulgaris did not present a latency phase, assuming exponential behavior immediately after inoculation. It obtained a maximum cell biomass of 1.9 g.L⁻¹, maximum specific growth rate of 0.51 d⁻¹, generation time of 1.35 d, and maximum biomass productivity of 0.36 g.L.d⁻¹.

Already under the heterotrophic regime, the microalgae showed a slight increase in the maximum cell concentration, reaching cell densities of 2.2 g.L⁻¹, as well as in the parameters of generation time (1.54 d) and maximum biomass productivity (0.42 g.L.d⁻¹), when compared to the photoautotrophic regime. In addition, the maximum specific growth rate parameter presented values lower than the autotrophic regime, around 0.45 d⁻¹.

To determine the optimal conditions for carotenoid production by microalgae, Figure 2 illustrates the interdependence between total carotenoid content and microalgae biomass productivity.

Lowercase letters indicate significant differences (p < 0.05) within the same column; uppercase letters indicate significant differences (p < 0.05) within the same column.

As observed in Figure 2, the general behavior of biomass production and carotenoid content presents a similar profile in both cultivation regimes, with carotenoid production being viable only if cultivation is carried out until the log and stationary phase due to biomass production. However, when the photoautotrophic cultivation regime is individually analyzed, it becomes clear that the trend in carotenoid content follows the exponential growth of biomass in all growth phases, with the final concentration of carotenoid content reaching its peak in the stationary phase. The same slope is observed for time zero and the lag phase in the heterotrophic regime. However, it is noted that in the log phase, there is a certain containment of the production of carotenoid content compared to the production of microalgal biomass when compared to the photoautotrophic regime.

In the heterotrophic regime, it is clear that there is a priority in directing the energy generated toward the formation of biomass (1.56 g.L⁻¹), reaching values slightly higher than photoautotrophic cultivation (1.46 g.L⁻¹). The carotenoid content, as it is a secondary compound, tends to be substantially reduced for the heterotrophic regime (604.79 µg.L⁻¹) when compared to the autotrophic regime (2921.70 µg.L⁻¹). This premise can be reinforced with the similar behavior presented in the stationary phase, with a maximum biomass concentration of 2.2 g.L⁻¹ for the heterotrophic regime and 1.9 g.L⁻¹ for the autotrophic regime. Indeed, the concentrations of carotenoid content differ substantially, with values in the order of 3866.48 and 1299.12 µg.L⁻¹ being found for the photoautotrophic and heterotrophic regimes, respectively.

According to Figure 3, it is possible to observe that the carotenoid blends present in the photoautotrophic regimes have high energy demands, with the maximum value identified in the stationary phase (9.60 kWh.L⁻¹) when compared to the heterotrophic regimes in the same condition (0.06 kWh.L⁻¹).

The disparity in the content and qualitative profile of carotenoids is notable since the photoautotrophic regime presents about 3 times greater quantity (~3866.48 µg.L⁻¹); this fraction is mostly composed of lutein (~30%) and β-carotene (~15%). Furthermore, in the adaptive (lag), exponential (log), and stationary phases, it is possible to identify the biosynthesis of unique carotenoids, such as all-trans-echinenone and 9-cis-echinenone, which have market prices estimated at up to 1250.00 USD/mg, resulting in a higher market value. On the other hand, when considering only the energy requirement, cultures in heterotrophic regimes, even with their lower productivity (1299.12 µg.L⁻¹), could be equivalent to the amount of carotenoids found in photoautotrophic regimes since three-batch cultures would consume about 0.18 kWh.L⁻¹, resulting in carotenoid content of up to 3897.36 µg.L⁻¹. Meanwhile, it is worth noting that around 20% of the costs of microalgae processing are related to the demand for nutrients and water, and, therefore, a more in-depth analysis must be established.

Similarly, when focusing on a specific carotenoid of interest, such as lutein, the scenario is viewed from a new perspective. In the photoautotrophic regime, the lutein concentration at time zero represents around 56% of the total content. However, as the biomass increases throughout the cultivation phases (lag, log, and stationary), its content decreases, reaching around 30%. The opposite is observed in the heterotrophic regime, where at zero time and the adaptive (lag) phase the content is 16 and 29%, respectively. However, as the cultivation phases progress, the content doubles, reaching values of ~61% in the stationary phase.

4. Discussion

Chlorella vulgaris exhibited a typical carotenoid profile of the Chlorophyta taxon, which mainly contains all-trans-lutein and all-trans-β-carotene [28]. Under light conditions, the main differences observed between the zero time and lag phase extracts were in the keto carotenoids. Notably, unique microalgae carotenoids such as all-trans-echinenone and 9-cis-echinenone, which were not detected at zero time, were formed during the lag phase of growth, with contents ranging from 114.53 to 112.78 μg.g⁻¹ (Table 2). These microalgae carotenoids are highly valued for their antioxidant potential, attributed to the 12 conjugated double bonds in their structure [29,30,31,32].

Considering the quantitative profile, it was observed that the total amount of carotenoids decreased from zero time (1849.88 μg.g⁻¹) until the microalgae reached the lag phase (1540.74 μg.g⁻¹) (Table 2). This behavior is due to the response of the main carotenoids all-trans-lutein (510.83 μg.g⁻¹) and all-trans-β-carotene (192.05 μg.g⁻¹) (Table 2) to the continuous illumination. Taking as reference the green algae (Chlorophyta), all-trans-lutein and all-trans-β-carotene also exhibited a similar behavior in the lag phase of Scenedesmus obliquus [33].

Based on the results, it has been suggested to employ high lighting levels, which may result in photo-inhibition. The high light intensity in low-biomass concentrations can lead to photo-inhibition, while low light levels in high-biomass density result in photo-limitation. These conditions affect lutein productivity. Lutein reduces reactive oxygen species formed under excess light, but high light intensity at early growth stages can damage photosynthetic complexes. Low light intensity is preferable early on to enhance lutein synthesis [34]. Chlorella vulgaris, a versatile and valuable organism commonly used for bioresource studies that determine the stoichiometric regulation of commercial carotenoid production, stands out both for its total carotenoid yield and for the variety of carotenoids it produces [10]. The carotenogenic profile of Chlorella can vary significantly; with modifications in cultivation, it is possible to stimulate the biosynthesis of special carotenoids [35].

Moreover, these changes are also observed in the different growth phases. For example, all-trans-luteoxanthin (27.90 μg.g⁻¹) was not detected at zero time and in the lag phase but was identified in the log phase (see Table 2). This indicates the formation of an epoxy compound (Figure 1), and its mechanism is well known [23].

Compared to the log phase, in the stationary phase except for all-trans-neoxanthin and 9-cis-lutein, all showed a significant difference (p < 0.05) (Table 2). Likewise, except for all-trans-zeaxanthin, all-trans-echinenone, 9-cis-echinenone, all-trans-α-carotene, all-trans-β-carotene, and 9-cis-β-carotene, all the other compounds were significantly (p < 0.05) higher in the stationary phase compared to the log phase.

As for the carotenoid profile, the experimental conditions of photoautotrophic growth may have contributed to changes. Among all the carotenoids identified at zero time, 9-cis-α-carotene (peak 20) was not detected during the growth stages. In contrast, in the growth stages, all-trans-echinenone and 9-cis-echinenone (peaks 17 and 18) were observed, whereas all-trans-luteoxanthin (peak 7) was only detected in the log and stationary phases.

Considering the carotenoid structures identified during photoautotrophic growth that were not present at zero time, the main reactions observed were ketolation and epoxidation (Figure 1). Already, the production of ketocarotenoids from all-trans-β-carotene (peak 21) involves the introduction of keto moieties at the 4,4′ position of the β-rings. These conversions of all-trans-β-carotene into all-trans-echinenone (one keto group) are carried out by the β-carotene ketolase [36,37,38,39]. Finally, one epoxide derivative of zeaxanthin (all-trans-luteoxanthin) was formed [23].

Thus, the carotenoids identified in this regimen play a crucial role in the metabolism of the microalga Chlorella vulgaris, contributing both to growth and overall cellular health. Furthermore, determining the optimal pathway for the production of lutein, β-carotene, and echinenone advances biotechnological efforts toward the development of targeted nutraceutical and pharmaceutical applications.

In the heterotrophic regime, qualitatively and quantitatively, the total carotenoid content was notably higher in the lag phase (1127.72 μg.g⁻¹) (see Table 2). The increase in pigment concentration during a short period of darkness has also been observed in other microalgae [40,41]. In that regard, the lag phase presented a carotenoid profile with eight different compounds compared to zero time. Of these compounds, one was the cis-isomer of all-trans-neochrome (peak 1), the cis-isomer of all-trans-neoxanthin (peak 2), the cis-isomer of all-trans-antheraxanthin (peak 6), 2′-dehydrodeoxymyxol (peak 15), 5,6-epoxy-β-carotene (peak 16), all-trans-echinenone (peak 17), and 9-cis-echinenone (peak 18). In contrast, 9-cis-α-carotene (peak 20) was only detected in the carotenoid extract at zero time. Considering these aspects, the heterotrophic strategy induced cyclization, ketolation, cis-isomerization, epoxidation, and epoxide-furanoxide rearrangement reactions (Figure 1).

The most abundant fraction of carotenoids in this regime corresponded to all-trans-lutein and its cis-isomers at 68.87% (stationary phase). This fact suggests that (in dark conditions) this growth phase is a potential source of this xanthophyll. Also, according to our results, [42] showed that with distinct culture media, such as cultures in heterotrophic [43] or mixotrophic systems [44,45], the production of lutein is substantially significant and may be considered as a potential source of commercial output of this pigment.

Although unique carotenoids (peaks 15, 17, and 18) were produced (lag phase), in general, the reduction in both the qualitative and quantitative content of carotenoids in the heterotrophic regime can significantly impact the overall value of microalgal biomass, as various carotenoids may be diminished or absent, potentially compromising the economic viability of production and utilization pathways [46]. However, by addressing certain aspects and implementing appropriate production strategies, it can still remain useful (see Section 3.3).

Microalgae biomass is the chassis of almost all microalgae bioproducts. Like carotenoid content, most microalgae bioproducts are intracellular; therefore, the high biomass productivity reflects their proportionality to the high production of target bioproducts. As a result, the kinetic growth parameters for Chlorella vulgaris under a photoautotrophic and heterotrophic growth regime were determined (Table 3).

During the determination of these parameters, the absence of the lag phase observed in photoautotrophic cultivation may result from the microalga’s rapid adaptation to the cultivation regime, as it is commonly maintained and propagated under similar conditions and can assimilate nutrients from the environment immediately [47].

In the heterotrophic regime, among all the kinetic parameters evaluated, the specific growth rate was the only one found to be lower compared to growth under light conditions. This is mainly due to the latency phase experienced in the first 24 h of hydraulic retention time. In addition, this behavior presented by the microalgae strain is associated with the adaptation of the photosynthetic apparatus to light deprivation conditions, as well as the disparity in time for the assimilation of the organic substrate. It is well known that species belonging to the phylum Chlorophyta have, as their main metabolic pathway, the assimilation of inorganic carbon (carbon dioxide). In this way, its photosynthetic apparatus relies on the presence of auxiliary enzymes, such as carbonic anhydrase, which promote greater access to inorganic carbon in the form of bicarbonate ions, accelerating the nutrient absorption process in the first hours of cultivation and, consequently, increasing the maximum growth rate and reducing the cell generation time [48].

Concurrently, expatiating on the kinetic results independently, considering only the final biomass density, is not enough to define the best scenario for the production of microalgae pigments. Thus, it was shown that the interdependence of total carotenoid content and microalgae productivity in both growth regimes (Figure 2) may be associated with biochemical mechanisms intrinsically selected by microalgae depending on cultivation regimes. Since carotenoids help capture energy from light, contributing to the formation of adenosine triphosphate (ATP), a primordial compound for the formation of biomass, it is suggested that during cell growth, the carotenoid content will not be prejudiced, depending on the supply of light energy during the photoautotrophic regime.

Considering the substantial differences in carotenoid content between the photoautotrophic (3866.48 µg.L⁻¹) and heterotrophic (1299.12 µg.L⁻¹) regimes, it can be stated that in photoautotrophic conditions, the energy generated, when present in adequate amounts, is simultaneously used for the biosynthesis of both biomass and secondary compounds, such as carotenoids. In contrast, in the heterotrophic regime, due to light deprivation, energy is primarily allocated to the formation of structural compounds, such as carbohydrates and proteins. However, studies have reported that an increase in the carbon source, preferably glucose, can increase carotenogenesis in microalgae species and, consequently, increase the production of carotenoids [49,50,51].

Given the aspects presented, arguably the most successful technological route for the voluminous carotenoid production is the photoautotrophic regime. However, it must be considered that microalgae biotechnological processes present numerous bottlenecks to be overcome, the main hotspot being energy demand, which is responsible for more than 80% of the process costs. Therefore, when prioritizing the photoautotrophic regime, the demands of artificial lighting rates, associated with the downstream steps, can further leverage the unfeasibility of microalgae-based processes. In this sense, the energy demands (electricity) necessary for the production of the carotenoid blend, as well as the production of lutein, the majority compound found in all phases and cultivation regimes, to establish a trade-off in the biotechnological process needed to be considered (see Figure 3).

Considering the dynamics of energy demands to obtain the blend of pigments, the production of carotenoids with low processing demands and lower commercial value can be explored for high-volume, low-cost applications, such as ingredients for animal feed and food purposes (heterotrophic conditions). Conversely, cultures under a photoautotrophic regime could be directed to the cosmetic, medical, and chemical bioindustries, since these specific biocompounds of interest must have a high biological value, once the sales prices compensate for the onerous energy requirements.

In parallel, when a single carotenoid of interest, such as lutein, is evaluated, the scenario gains a new vision. Since photoautotrophic cultures have high energy expenditure and still have higher maximum rates of cell growth, the heterotrophic regime is suggested as the alternative that best provides viability in terms of lutein production.

Finally, from an overview of the process, the photoautotrophic regime conducted until the adaptive (lag) phase is what provides the best balance between the variables analyzed, as it allows for the use of both the carotenoid blends with high market value, as well as presenting a satisfactory lutein content, under a moderate energy demand.

5. Conclusions

The results in the present study demonstrated that, in general, carotenoid production is only viable if cultures are carried out until the log and stationary phase for both regimes (photoautotrophic and heterotrophic). Therefore, it is concluded that a high biomass yield is essential to ensure a high pigment yield. Additionally, the best condition for producing a blend of carotenoids and specific carotenoids, such as lutein, was observed under the photoautotrophic regime, carried out until the exponential (log) phase. On the other hand, when the focus is on singular lutein production, cultures carried out until the stationary phase under heterotrophic regimes showed the best performance. Thus, the scientific advances found in this study will assist in process control through decision-making about the best biochemical route for the particular production of compounds of interest, depending on the target industry and interest of the chosen bioproduct.

6. Future Perspectives

Future research could focus on consolidating processes at the industrial level, with more in-depth evaluations of process scalability and economic viability. The results of this study also highlight the need for further studies to obtain additional microalgae compounds, not just for carotenoid production. A key objective for future research is to analyze the applicability of microalgae-based carotenoids in food matrices, as well as the bioavailability and mechanisms of action of these compounds at the metabolic level.