Productivity and Carbon Utilization of Three Green Microalgae Strains with High Biotechnological Potential Cultivated in Flat-Panel Photobioreactors

Abstract

Microalgae biotechnology is increasingly applied across diverse fields, from food and medicine to energy and environmental protection, with strain selection being crucial for both target product accumulation and scalability potential. In this study, we for the first time assess the scalability of two new promising green microalgae strains, Neochlorella semenenkoi IPPAS C-1210 and Desmodesmus armatus ARC-06, in 5-L flat-panel photobioreactors. The growth characteristics of each culture, along with their biochemical composition and CO₂ utilization efficiency, were examined and compared to the well-studied model strain Chlorella sorokiniana IPPAS C-1. While C-1 achieved the highest biomass concentration (7.1 ± 0.4 g DW L⁻¹ by day 8) and demonstrated superior specific productivity (1.5 ± 0.1 g DW L⁻¹ d⁻¹) and CO₂ utilization efficiency (average 25.4%, peaking at 34% on day 3), ARC-06 accumulated the highest starch content (51% of DW), twice that of C-1. Strain C-1210 showed intermediate performance, reaching 6.8 ± 0.8 g DW L⁻¹ biomass with a CUE of 22.7%, whereas ARC-06 had the lowest CUE (12.8%). These results, combined with proposed cultivation optimization strategies, provide a foundation for scaling up N. semenenkoi and D. armatus production in industrial flat-panel PBR systems.

1. Introduction

Microalgae biotechnology gains increasing application in different fields, starting from such well-known areas as the food industry and medicine and expanding, but not limited to, energy and environmental protection [1,2,3,4]. During the pre-production stage, the most important step is to determine the potential of a certain microalgal strain both in terms of accumulation of target products and the possibility of scaling-up, including cost efficiency [5,6]. No biotechnological application could be achieved if scaling-up is impracticable or ineffective. To assess its potential for industrial cultivation, a microalgal strain that has been successfully cultivated in small vials (0.2–0.5 L) must be scaled up in laboratory reactors (2–20 L). Successful scaling-up has several important criteria: (1) achieving and maintaining high specific growth rates and biomass concentrations; (2) significant reduction of lag phase; (3) relatively simple maintenance and control of biomass production and target product accumulation processes; (4) resistance to contamination and insignificant influence of other negative growth factors [7].

Currently, only a few dozen microalgal strains are cultivated industrially. Among Chlorophyta, the most commonly used species belong to the genera Chlorella, Haematococcus, Desmodesmus, Dunaliella, Scenedesmus and Tetraselmis [8,9]. However, the vast majority of species remain largely unexplored. Identifying new strains with scaling potential is therefore crucial for advancing microalgae biotechnology.

For the present study, two strains of green algae were selected. The strain IPPAS C-1210 represents a recently described genus and species, Neochlorella semenenkoi [10]. It was reported to be a potential producer of protein, starch, and triacylglycerols and was characterized by thermotolerance, halotolerance, alkaliphily, and the ability to use a wide range of carbon and nitrogen sources [10,11,12]. However, further research is required to evaluate its potential for industrial biomass production, as its intensive cultivation in PBRs has not yet been examined.

The ARC-06 strain has been recently isolated and identified as Desmodesmus armatus [13] and has not been previously assessed for its biotechnological potential. Currently, strains from the Desmodesmus clade are used as producers of various polysaccharides [14], for wastewater treatment [15,16,17], and for bio-mitigation of CO₂ and other greenhouse gases [18].

In this work, we present the first scaling-up stage of intensive cultivation of these strains in laboratory photobioreactors. The growth characteristics of each culture, their biochemical composition (proteins, carbohydrates, and pigments), and CO₂ utilization efficiency were studied in a series of experiments and compared with the well-studied model strain Chlorella sorokiniana IPPAS C-1 [19,20,21]. The C-1 strain was also re-cultured in the present study to confirm the repeatability of cultivation results. These data are important for developing optimized cultivation regimes for the industrial production of N. semenenkoi IPPAS C-1210 and D. armatus ARC-06 in flat-panel PBRs.

2. Materials and Methods

2.1. Microalgae Strains and Maintenance Conditions

The axenic strain of C. sorokiniana IPPAS C-1 and N. semenenkoi IPPAS C-1210 were obtained from the Collection of Microalgae and Cyanobacteria IPPAS (K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia). The axenic cultures were maintained at 22 °C under continuous 2700–3000 K LED illumination (30 μmol photons m⁻² s⁻¹) on slants of Tamiya agar medium [20,22] in glass tubes and in Petri dishes with 3N Bold basal agar medium (BBM 3N) [23], respectively. For the experiments, cell cultures of C. sorokiniana and N. semenenkoi were grown for 10–14 days in 300-mL Erlenmeyer flasks, filled with 100 mL of ½ Tamiya modified medium [20] at 27 °C under continuous 5500–6500 K luminescent illumination (50 μmol photons m⁻² s⁻¹) in a growth chamber MLR-352-PE (Panasonic, Japan). ½ Tamiya modified medium composition, g L⁻¹: NaNO₃—2.1; MgSO₄ × 7H₂O—1.25; KH₂PO₄—0.625; FeSO₄ × 7H₂O—0.009; EDTA—0.037; trace element solution (TES) 1 mL L⁻¹; TES composition, g L⁻¹: H₃BO₃—2.86; MnCl₂ × 4H₂O—1.81; ZnSO₄·7H₂O—0.222, MoO₃ × 2H₂O—0.018, NH₄VO₃—0.023.

The axenic strain of Desmodesmus armatus ARC-06 was obtained and isolated as described in [13] from the Yenisei Gulfs (Siberia). The axenic culture was maintained in Petri dishes with BBM-3N agar medium at 22 °C under continuous 2700–3000 K LED illumination (30 μmol photons m⁻² s⁻¹). For the experiments, D. armatus was cultivated for 10–14 days in 300 mL Erlenmeyer flasks filled with 100 mL of BBM 3N, placed into an orbital shaker, at 22 °C and under continuous 2700–3000 K LED illumination averaging 50 μmol photons m⁻² s⁻¹.

2.2. Algae Pre-Culture for Photobioreactor Inoculation

Intensive cultivation in PBRs under high CO₂ and light requires higher temperatures. This necessitates a gradual adaptation process for collection strains that are maintained at lower temperatures and light conditions. The stepwise scaling-up process is illustrated in Figure S1.

The algae inoculum was grown aseptically in the vessels of the laboratory system for intensive cultivation as described in [21]. C. sorokiniana IPPAS C-1 and N. semenenkoi IPPAS C-1210 were grown for four days in ½ Tamiya medium at 32.0 ± 0.6 °C under continuous 3000 K LED illumination of 500 μmol photons m⁻² s⁻¹ at the start and increased to 900 μmol photons m⁻² s⁻¹ 1–2 days before inoculation in PBRs (the description of the LED module and the spectral composition of the light are presented in the Supplementary Material in Figure S2). D. armatus ARC-06 was initially cultivated under the same conditions as C-1 and C-1210 but in BBM 3N medium. Subsequent rounds employed modified parameters: 27.0 ± 0.5 °C and continuous 3000 K LED illumination (300 μmol photons m⁻² s⁻¹).

Algae culture mixing and aeration were achieved by bubbling with a sterile gas-air mixture (GAM) containing 1.5–2% CO₂. The resulting culture with a dry biomass concentration of 2–5 g DW L⁻¹ was used as inoculum for the next stage. The contents of vessels were mixed in a sterile graduated cylinder, from which the required volume was aseptically transferred to the reactor using a peristaltic pump. The inoculum volume was adjusted to achieve an initial culture density of 0.1–0.2 g DW L⁻¹ for each strain in each PBR. Inoculum parameters (OD₇₅₀, pH, volume, and biomass concentration) are presented in Table S1.

2.3. Flat-Panel Photobioreactors

The photobioreactor (PBR), with a 5-L working volume (FP-5 PBR), consists of a glass aquarium with an internal volume of 361 mm × 40 mm × 460 mm, submersible module with cooling and hot water supply systems, and two LED modules, consisting of 18 rows of LED strips, similar to that used for algae pre-culture (Figure S2). Three PBRs of this type have been used in the current work as three biological replicates. Principal experimental design and detailed PBR design were described earlier [19].

The cultivation system featured an adjustable LED illumination setup with programmable irradiance control (0–100% output range). At full power (100%), the system delivered an average irradiance of 800 ± 70 μmol photons m⁻² s⁻¹ across the PBR’s light-receiving surface. The LED modules were mounted flush against the PBR sidewalls to ensure direct and uniform light exposure to the culture. The optimal conditions for intensive cultivation of C. sorokiniana IPPAS C-1 in FP-5 PBR were determined before [19]: GAM flow, 1 L min⁻¹ (0.2 vvm); CO₂ concentration, 1.5% ± 0.04; illumination level, 800 ± 70 μmol photons m⁻² s⁻¹; temperature, 36.0 ± 0.5 °C. This combination was applied for two other strains, but the described conditions were not suitable for D. armatus ARC-06 cultivation. First trial with this strain was unsuccessful; the lag phase lasted for almost two days; the linear phase was characterized by a large variability in biomass accumulation rate. The ARC-06 strain was restarted under 27 ± 0.5 °C and 270 ± 15 μmol photons m⁻² s⁻¹ as described in [13,17,24,25]. On the 6th day, the illumination level was increased twofold up to 545 ± 35 μmol photons m⁻² s⁻¹.

The illumination level (μmol photons m⁻² s⁻¹) in the working volume of the PBRs and on the surface of the LED modules was measured with a quantum meter LI-189 equipped with LI-190SA quantum sensor (LI-COR, Lincoln, NE, USA). Spectral composition of the LED light was measured by the LI-180 spectrometer (LI-COR, Lincoln, NE, USA). The average irradiance level has been calculated after a series of measurements on the PBR inner surface.

In all experiments and during pre-cultivation, water was purified by a six-stage reverse osmosis system AP-800DIR-400 (AquaPro Industrial Co., Ltd., Nanking E. Rd., Taipei, Taiwan) with final treatment by UV flow filter VIQUA VT4/2 BWT (Viqua, West Guelph, ON, Canada). All containers, hoses, filters, and liquids were either sterilized by autoclaving or treated with hot steam and 70% ethanol.

2.4. Gas-Air Mixture Supply

The GAM was supplied to the algae suspension through an aquarium sprayer, Hailea HL-AC04, 252 holes in total (6 holes in circumference with a step of 10 mm and Ø 1 mm), placed at the bottom of each PBR. The sprayer length was 300 mm. Pure CO₂ from a cylinder was mixed with air by a compressor in a mixing unit. The GAM was then passing through rotameters, CO₂ concentration sensors and filters (Vent Filter Hydrophilic PTFE 0.45 μm Hawach Scientific, Xi’an City, China), ending in sprayers that released it eventually into the suspension. The GAM volumetric flow rate was 1 L min⁻¹ in all experiments. Concentration of CO₂ and the R_GAM were controlled by rotameters. In previous studies [19], the GAM supply conditions for C. sorokiniana IPPAS C-1 cultivation in the same PBRs have been optimized and used in the current work.

2.5. Temperature Control System

Thin-walled stainless-tube heat exchangers were installed perpendicular to the suspension flow in the PBRs. The suspension temperature was controlled due to the periodic flow of the coolant through the heat exchanger tube. The automatic regulation of the cultivation temperature was controlled by a signal from a temperature sensor located in the internal volume of each PBR.

The temperature conditions in experiments with C. sorokiniana IPPAS C-1 and N. semenenkoi IPPAS C-1210 were the same (36.0 ± 0.5 °C), for D. armatus ARC-06, 27.0 ± 0.5 °C. The temperature regime for C-1 and C-1210 was set in accordance with the high growth rates of these strains during the first three days in laboratory vessels [21] and in PBRs [19]. The temperature for ARC-06 cultivation referred to published data [17,24,25].

2.6. Growth Characteristics

Growth parameters were calculated as described before [20]. The optical density of the suspension was measured at 750 nm by the Genesys 10S UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA). The samples with highly concentrated culture were diluted to avoid ‘saturation’ of OD measurements that result when insufficient light reaches the photodetector [26]

To measure the dry weight, 1–10 mL samples were precipitated by centrifugation, washed with distilled water, and dried at 80 °C overnight in pre-weighed plastic microtubes in a heat oven. The dry weight of each sample was measured in triplicate.

The following parameters were used to estimate the growth rate:

• The productivity (P) was estimated by dry weight (g DW L⁻¹ d⁻¹):

$$P=\left({\rho }_2-{\rho }_1\right)/\left(t_2-t_1\right)$$

(1)

where ρ₁ and ρ₂ are biomass concentrations measured at time 1 (t₁) and time 2 (t₂).

Using the correlation coefficient (k) between OD₇₅₀ and biomass concentration, the following equation was applied for the points without direct DW measurement:

$$P=k·\left({OD750}_2-{OD750}_1\right)/\left(t_2-t_1\right)$$

(2)

where OD750₁ and OD750₂ are the optical densities of the culture at 750 nm measured at time 1 (t₁) and time 2 (t₂). Correlation coefficients of dry weight (DW) content and optical density were calculated based on DW measurements at different OD₇₅₀ levels (Figure S3).

• The specific growth rate (μ) was estimated by the change in the culture OD (d⁻¹):

$$\mu =\ln \left({OD750}_2/{OD750}_1\right)/\left(t_2-t_1\right)$$

(3)

• The total biomass productivity of each strain was calculated as the difference between final and initial biomass concentrations divided by the total cultivation period.

2.7. pH Measurements

The pH level of every sample was measured by a Mettler Toledo SevenEasy pH meter equipped with an Inlab 413 electrode. Measured pH levels are presented in Supplementary Materials (Figure S4).

2.8. Carbon Dioxide Utilization Efficiency

The carbon dioxide utilization efficiency (CUE) was calculated as described in [19] (%):

$$CUE=1.88·(M_{PBR}-M_0)/M_{CO2}·100$$

(4)

where 1.88 is a coefficient to recalculate the amount of fixed CO₂ in a microalgae cell, based on a typical microalgal molecular formula, CO_0.48H_1.83N_0.11P_0.01 [27]; (M_PBR − M₀)—accumulated weight of dry biomass in the working volume of PBR, and M_CO2 is the mass of CO₂ that was supplied through the PBR. M_CO2 was calculated as total CO₂ volume multiplied by ρ_CO2 = 1.8 g L⁻¹ (28–30 °C, 10⁵ Pa). CUE is the ratio of the total supplied CO₂ converted into biomass that depends not only on the capability of an algal strain, but also on the design parameters of a PBR.

Total CO₂ volume (V_CO2) was derived from rotameters (flow rate R_GAM) and gas analyzer indications (n_CO2).

$$V_{CO2}=n_{CO2}·R_{GAM}·\left(t_2-t_1\right)$$

(5)

2.9. Biochemical Composition

Algae suspension sub-samples (0.5–5 mL) were precipitated by centrifugation, stored at −20 °C, and analyzed for protein, starch, and pigment content. The detailed procedures of protein and starch extraction and measuring their concentrations are described in [11].

Briefly, to extract and estimate the total protein, the frozen cells were mechanically disrupted with glass beads in methanol at 30 kHz for 10 min using a vibration mill (Retsch MM 400, Haan, Germany). Then, disrupted cells were repeatedly washed with methanol until colorless to minimize spectral interference. After centrifugation, methanol was discarded, and the pellet was dried at 37 °C. Total proteins were extracted by incubating the dried precipitate in extraction buffer (50 mM Tris-HCl, pH 6.8; 10 mM EDTA, 2% SDS) at 95 °C for 10 min. Following debris removal via centrifugation, protein concentration was quantified using the bicinchoninic acid (BCA) assay with BSA standards.

For estimating storage polysaccharides, the frozen cell pellets were treated with 400 μL of 30% KOH at 95 °C for 90 min to dissolve the storage polysaccharides and hydrolyze smaller sugars. The polysaccharides were then ethanol-precipitated at –20 °C overnight, collected via centrifugation, and hydrolyzed to glucose using 2 M HCl (80 μL, 95 °C, 30 min). After neutralization (2 M NaOH, phosphate buffer), glucose concentration was measured using the phenol-sulphuric method with glucose as a standard. The obtained results primarily reflected storage carbohydrates (starch), though minor contributions from cell wall polysaccharides were possible.

To measure pigment content (as described in [11]), the frozen microalgae cells were disrupted with glass beads in methanol at 30 kHz for 10 min using a vibration mill (Retsch MM 400, Germany). After debris removal by centrifugation, the absorption spectra (350–750 nm) of the methanol extracts were obtained using a Cary 300 double-beam spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The concentration of pigments was estimated using the formulas described in [28,29]:

$$\left[Chl a\right]= -8.0962·\left(A_{652}-A_{720}\right)+16.52·\left(A_{665}-A_{720}\right)$$

(6)

$$\left[Chl b\right]=27.44·\left(A_{652}-A_{720}\right)-12.17·\left(A_{665}-A_{720}\right)$$

(7)

$$\left[Car\right]={\displaystyle \frac{1000·\left(A_{470}-A_{720}\right)-2.86·Chl a-129.2·Chl b}{221}}$$

(8)

where A_xxx—light absorption at a wavelength of xxx nm,

• [Chl a]—chlorophyll a content,
• [Chl b]—chlorophyll b content,
• [Car]—total carotenoid content.

All measurements were carried out in triplicate.

2.10. Algae Biomass Post-Processing and Storage

After cultivation, the suspension was peristaltically pumped (VT600-2J pump, Longer Precision Pump Co., Baoding, China) into a tubular centrifuge (GQ-75, Eurasia group, Russia-China) and centrifuged at 12,000 rpm for 1.5–2.0 h to separate the cells from the liquid phase. The resulting concentrated biomass paste, with a humidity of 65–75%, was placed in Petri dishes and frozen at −70 °C (MDF-U3386S, Panasonic Sanyo, Osaka, Japan). Next, the samples were lyophilized for 24 h at 100 Pa (FreeZone 6 L, Labconco, Kansas City, MO, USA). The residual moisture content did not exceed 1.0%. The dried biomass was weighed and stored in airtight containers in a dark place at room temperature.

2.11. Statistics

The growth curves and biochemical composition of the cells are represented as mean values for three biological replicates ± SD.

3. Results

3.1. Growth Characteristics of Algae Under Intensive Cultivation Regime

The growth characteristics of the batch cultures are presented in Figure 1 with corresponding visible biomass accumulation in PBRs shown in Figure S5. Detailed growth parameters and productivity metrics are presented in

Table 1. Results of batch cultivation of C. sorokiniana IPPAS C-1 and N. semenenkoi IPPAS C-1210 cultivated in laboratory flat-panel photobioreactors under similar conditions: ½ Tamiya medium, I_ave = 800 ± 70 µmol photons m⁻² s⁻¹, T = 36.0 ± 0.5 °C, R_GAM = 0.2 vvm and n_CO2 = 1.5%.

Parameters	Chlorella sorokiniana C-1	Neochlorella semenenkoi C-1210
Starting biomass concentration, g DW L−1	0.15 ± 0.01	0.11 ± 0.02
Biomass concentration at the 3rd day, g DW L−1	4.60 ± 0.24	3.73 ± 0.73
Final biomass concentration (day 8), g DW L−1	7.10 ± 0.41	6.76 ± 0.84
Specific growth rate (max), day−1	2.23 ± 0.05	2.32 ± 0.36
pH starting level	6.28 ± 0.01	6.29 ± 0.06
pH maximum level	8.33 ± 0.03	8.34 ± 0.09
The productivity during days 0–3, g DW L−1 d−1	1.48 ± 0.08	1.20 ± 0.25
Total productivity (8 days), g DW L−1 d−1	0.87 ± 0.05	0.80 ± 0.06
Total biomass yield, g DW	34.73 ± 2.02	33.22 ± 4.22

and

Table 2. Results of batch cultivation of D. armatus ARC-06 cultivated in laboratory flat-panel photobioreactors under conditions: in BBM N3, I_ave = 270 ± 15 → 545 ± 35 µmol photons m⁻² s⁻¹, T = 27.0 ± 0.5 °C. R_GAM = 0.2 vvm and n_CO2 = 1.5%.

Parameters	Desmodesmus armatus ARC-06
Starting biomass concentration, g DW L−1	0.15 ± 0.01
Biomass concentration at the 3rd day, g DW L−1	2.10 ± 0.03
Final biomass concentration (day 8), g DW L−1	4.96 ± 0.30
Specific growth rate (max), day−1	1.00 ± 0.05
pH starting level	6.74 ± 0.03
pH maximum level	7.99 ± 0.06
The productivity during days 0–3, g DW L−1 d−1	0.68 ± 0.01
Total productivity (8 days), g DW L−1 d−1	0.61 ± 0.04
Total biomass yield, g DW	24.05 ± 1.46

.

The strains C-1 and C-1210 exhibited immediate exponential growth without a lag phase, whereas ARC-06 had a one-day lag phase before growth initiation. The C-1210 strain had the highest specific growth rate of 2.32 ± 0.36 d⁻¹ on the first day of cultivation. The specific growth rates of strains C-1 and C-1210 showed a progressive decline from the second day of cultivation, while the decline of the ARC-06 specific growth rate became apparent on the third day (Figure 1). Beyond day 6 of cultivation, further biomass accumulation of all strains became energetically unfavorable, with diminishing returns on productivity relative to cultivation time and resource inputs. The strains C-1 and C-1210 had maximal specific productivity P during the first 3 days of cultivation, 1.48 ± 0.08 g DW L⁻¹ d⁻¹ and 1.20 ± 0.25 g DW L⁻¹ d⁻¹, respectively. The maximal specific productivity of ARC-06 was lower (P = 0.72 ± 0.06 g DW L⁻¹ d⁻¹). The maximal biomass concentration of 7.10 ± 0.41 g DW L⁻¹ was reached at the 8th day of cultivation for the C-1 strain.

The pH level in the C-1 and C-1210 cultures increased from 6.3 (day 0) to 8.2–8.3 (day 3), remained stable at this level until the end of the experiment (day 8). Similarly, the pH level for ARC-06 has increased from 6.7 (day 0) to 7.8 (day 3) and remained stable until the end of the experiment (Figure S4).

The strains C-1 and C-1210 were characterized by a similar range of the total biomass yield (33–35 g DW per 5 L of suspension). Total biomass yield of strain ARC-06 was 25% lower than that of strains C-1 and C-1210 (24.05 g DW per 5 L of suspension). Finally, biomass was lyophilized. The appearance of the biomass obtained as a result of centrifugation and lyophilization stages is presented in the Supplementary Materials (Figure S6). The DW biomass loss through all post-processing stages was 5–10%.

The CO₂ utilization efficiency and the daily biomass increase are presented in Figure 2. CUE levels remain stable in growing cultures for almost 3–4 days. Among all strains, C-1 demonstrated the highest CUE of 34.01 ± 1.8% on the 3rd day of cultivation (Figure 2a). Obviously, CUE might be higher on the day with the highest daily biomass increase (Figure 2b).

3.2. Protein and Carbohydrate Composition of the Algae

Strains of Chlorella and Desmodesmus green algae serve in biotechnology as sources of starch and proteins, so the content of these compounds was estimated in the cell culture (Figure 3a). The cells of ARC-06 had almost twofold higher starch content (51.7%) than the cells of C-1 and C-1210 (22.5% and 29.5%, respectively). An opposite pattern was observed for the protein content, when it was three times higher in the cells of strain C-1 compared to ARC-06 (22.2% and 6.46%, respectively). At first glance, the contents of protein and starch were inversely related to each other in cells of C-1210 and ARC06. However, cells of the strain C-1 had a balanced composition of proteins and carbohydrates, 22.2% DW and 22.5% DW, respectively.

3.3. Pigment Content

The pigment content after 8 days of cultivation is presented in Figure 3b. The highest content of chlorophyll a (26 mg g DW⁻¹) and chlorophyll b (7.8 mg g DW⁻¹) was found in the cells of the strain C-1; the strain ARC-06 exhibited similar chlorophyll content but had the highest content of carotenoids (8.9 mg gDW⁻¹). The lowest pigment content was found in the cells of the strain C-1210.

4. Discussion

4.1. Intensive Cultivation in Flat-Panel Photobioreactors

Earlier, we cultivated C. sorokiniana IPPAS C-1 during a shorter period of 3–4 days [19]. Compared to previous runs, the productivity, specific growth rates and CUE of C. sorokiniana IPPAS C-1 remained nearly identical. During the first three days of cultivation in FP-5 PBRs, they showed only minor variations (2%, 1% and 3%, respectively), demonstrating high experimental reproducibility. In long-term batch mode, the maximum biomass concentration (7.10 ± 0.41 g DW L⁻¹) was achieved by day 8. Another strain of C. sorokiniana (AM-02) reached a maximum biomass concentration of 3.08 ± 0.31 g DW L⁻¹ after 5 days of cultivation [30]. This value is only half of the biomass concentration of our IPPAS C-1 strain achieved after the same 5-day period (6.26 ± 0.48 g DW L⁻¹). We attribute this significant difference to several factors: the use of rich modified ½ Tamiya medium in our study versus BBM N3 medium [30], and different photobioreactor designs—flat-panel PBRs in our study compared to the cylindrical fermenter-type reactor (Labfors 4 Lux photobioreactor) used in [30]. The reactor geometry likely affects light absorption efficiency [31], and cells in flat-panel PBRs in our experiments probably had more available light, despite the 30% higher illumination levels employed in [30].

Previous studies have reported high specific growth rates for N. semenenkoi IPPAS C-1210, ranging from 2.610 d⁻¹ [11] to 3.024 d⁻¹ [21]. However, these experiments were conducted in small laboratory-scale cylindrical vessels (0.20–0.25 L) [10,11,21]. In our study, strain C-1210 maintained a similarly high specific growth rate (2.32 d⁻¹)—more than double the 0.92 d⁻¹ rate observed when this strain was used for wastewater treatment [32]. This discrepancy likely results from differences in cultivation conditions (media composition, temperature, CO₂ concentration in the GAM, illumination levels, etc.). Our results demonstrate that a 20-fold scale-up does not significantly impair the growth rate of N. semenenkoi IPPAS C-1210. Notably, despite exhibiting a 26.5% lower CUE, this strain achieved comparable growth rates and final biomass concentrations to C. sorokiniana IPPAS C-1, suggesting strong potential for industrial-scale cultivation.

Compared to other studied Desmodesmus strains, our strain ARC06 exhibited competitive growth characteristics. The strain Desmodesmus sp. VIT was cultivated in batch mode using 2-L bag photobioreactors under three illumination levels (8000, 16,000, and 32,000 Lux, corresponding to I_ave = 110, 220, and 440 μmol photons m⁻² s⁻¹, respectively) and four light:dark regimes (12:12, 16:8, 20:4, and 24:0 h) [33]. The maximum biomass concentration (1.0 g DW L⁻¹) was achieved at the highest irradiance (440 μmol photons m⁻² s⁻¹). Under continuous illumination (24:0 h) at 220 and 440 μmol photons m⁻² s⁻¹, the results were comparable to ours due to similar conditions. However, at equivalent irradiance (440 μmol photons m⁻² s⁻¹), the reported biomass productivity (57.2 ± 3.1 mg DW L⁻¹ d⁻¹) [33] was an order of magnitude lower than our results (606 ± 37 mg DW L⁻¹ d⁻¹).

Another Desmodesmus strain, D. insignis (former Scenedesmus quadricauda var. insignis) JNU24, was cultivated in BG-11 medium with varying initial NaNO₃ concentrations and in diluted dairy wastewater (DWW; four concentrations) to assess growth performance, starch production, and nutrient uptake capacity [16]. After 21 days in 24-L flat-plate bioreactors (30 mm light path, two-sided illumination at 300 μmol photons m⁻² s⁻¹, 24.0 ± 1.0 °C), the maximum biomass (9.75 g DW L⁻¹), starch content (4.75 g DW L⁻¹), and productivity (230 mg L⁻¹ d⁻¹) were achieved in 75% DWW—approximately double the values obtained in BG-11 medium. In our study, D. armatus ARC-06 reached 4.96 ± 0.30 g DW L⁻¹ by day 8, exceeding the ~4 g DW L⁻¹ reported for D. insignis at comparable time points [16]. When grown in column-type PBRs, D. insignis showed medium-dependent biomass concentrations: 4.00–6.23 g DW L⁻¹ in BG-11 versus 6.0–8.0 g DW L⁻¹ in DWW [16].

Additional evidence supporting the biotechnological potential of Desmodesmus strains comes from studies [17,24]. In [17], the locally isolated Desmodesmus armatus (formerly Scenedesmus hystrix var. armatus) MO1 demonstrated efficient nutrient removal from municipal wastewater, achieving 1.375 g DW L⁻¹ biomass with high protein content and excellent nitrogen/phosphorus uptake after 8 days. The relatively low biomass yield likely resulted from suboptimal light conditions (5000 Lux ~ 70 μmol photons m⁻² s⁻¹) and lack of CO₂ supplementation. Study [24] examined three Desmodesmus strains isolated from White Sea benthic invertebrates: strain 3Dp86E_1 showed exceptional CO₂ tolerance (up to 100%), strain 1Hp86E_2 reached 3.0 g DW L⁻¹ by day 10, and strain 1Pm66B had relatively high lipid content (~220 mg g DW⁻¹).

These findings demonstrate that Desmodesmus strains exhibit dual tolerance to both elevated CO₂ concentrations in GAM and high nitrate levels (e.g., in DWW). This physiological adaptability, combined with their ability to thrive in low-cost growth media, makes them particularly suitable for large-scale environmental applications. Our future research will focus on further characterization of these capabilities in the ARC-06 strain through targeted cultivation experiments, including optimization of growth conditions to improve CUE, which was approximately 50% lower than that of C-1 in the present study.

Enhancing microalgae productivity in FP-5 photobioreactors presents significant challenges, primarily requiring precise optimization of cultivation conditions through automated measurement and control systems. Furthermore, successful industrial-scale cultivation demands strict consistency across all operational parameters, particularly in maintaining standardized photobioreactor geometry (including both shape and dimensions), to maximize biomass yields within reduced production timeframes.

4.2. Biochemical Composition of Cells

The cells of Chlorella sorokiniana IPPAS C-1 showed nearly equal relative content of proteins and starch at the cultivation endpoint. However, our previous experiments with this strain demonstrated twice the protein content (up to 38 ± 8.6% DW) while maintaining similar starch levels (20 ± 0.6% DW) [19]. Notably, both experimental series used identical cultivation conditions and FP-5 photobioreactors, differing only in duration (3 days versus 8 days in the current study). This discrepancy likely reflects known physiological patterns, as cells typically accumulate maximal protein content during the exponential growth phase [11]. In the present work, C. sorokiniana IPPAS C-1 contained 33.77 mg g DW⁻¹ total chlorophylls and 6.27 mg g DW⁻¹ carotenoids—values comparable to those reported for Chlorella sorokiniana AM02 (30.62 mg g DW⁻¹ and 5.09 mg g DW⁻¹, respectively) [30].

Low pigment content in Neochlorella semenenkoi C-1210 cells during stationary growth phase (days 8–9) was previously reported, with values of ~9 mg g DW⁻¹ for Chl a, ~2 mg g DW⁻¹ for Chl b, and ~3 mg g DW⁻¹ for carotenoids [11]—approximately double what we observed in the current study (4.8 ± 0.5, 1.3 ± 0.1, and 2.3 ± 0.2 mg g DW⁻¹, respectively). The C-1210 strain showed higher starch content than Chlorella sorokiniana C-1 but lower than Desmodesmus armatus ARC-06. This elevated starch accumulation in C-1210 correlated with significantly reduced total protein content (9.5 ± 1.7% DW) compared to C-1 (22.2 ± 1.0% DW). Previous reports of stationary-phase C-1210 cells showed 18% DW protein and 12% DW carbohydrates [10], aligning with our experimental range. Cellular biochemical composition varies significantly with growth phase and cultivation conditions. During the first 3 days of cultivation, C-1210 exhibited substantially higher pigment content (~36 mg g DW⁻¹ Chl a; 8.8 mg g DW⁻¹ each for Chl b and carotenoids) [10], with similar patterns observed for proteins, lipids, and carbohydrates. These findings highlight the importance of optimizing C-1210 cultivation regimes for targeted compound production.

Our studies indicate that Desmodesmus armatus ARC-06 shows promising potential for starch production. Other researchers [33] reported comparable carbohydrate levels (25.4 ± 0.7% DW) in Desmodesmus sp. VIT under similar light intensity but with a 16:8 h light:dark cycle, while continuous illumination (24 h) yielded lower values (17.9 ± 0.7% DW). This significant difference (approximately half of our measured values) likely results from their cultivation without CO₂ supplementation, unlike our CO₂-enriched system. Previous work [16] documented maximal starch (39.42% DW) and protein (32.53% DW) accumulation in Desmodesmus strains by day 9 when grown in BG-11 medium using flat-panel photobioreactors. Relative to our findings, this represents 1.31-fold lower starch content but substantially greater protein accumulation (over fivefold higher than our 6.46 ± 0.46% DW). Notably, dairy wastewater cultivation achieved 48.75% DW starch by day 21 [16], with concurrent protein and total carbohydrate contents of 19.77% and 61.90% DW, respectively. Even higher protein levels (46.14% DW) were reported for D. armatus MO1 [17], with wastewater-cultivated cells containing 20.25% DW carbohydrates. Consistent with other observations, protein and starch contents exhibited an inverse correlation.

In our experiments, the total chlorophyll and carotenoid content in Desmodesmus armatus ARC-06 cells measured 28.17 mg g DW⁻¹ and 8.09 mg g DW⁻¹, respectively—approximately 2.5-fold lower than the maximum values reported for Desmodesmus sp. 1Pm66B (~70 mg g DW⁻¹ and ~30 mg g DW⁻¹) [24]. The same study [24] demonstrated that pigment content undergoes significant variation under nitrogen-limited cultivation conditions.

4.3. Application Prospects

Cultivation of different high-productivity strains with a variety of biochemical content opens opportunities to use biomass and valuable compounds in various industries. The three studied strains of green microalgae are rich in nutrients, grow fast, and have certain tolerance to water environmental pollutants and contamination [4,15,16,17,18,24,32]. These strains represent promising sources of natural pigments, including chlorophyll and carotenoids. Chlorophyll derivatives serve as natural food colorants and cosmetic components with demonstrated antioxidant and anticancer properties [34,35,36,37,38]. Certain microalgae strains are already widely used in industry as sources of carotenes and xanthophylls [39]. Due to their broad spectrum of beneficial effects on human and animal health, xanthophylls are actively utilized in the food, feed, and pharmaceutical industries. Numerous studies confirm their biological activity, including protection against oxidative stress and positive modulation of metabolic processes [40,41,42,43,44].

As sustainable starch producers, microalgae outperform traditional crops like corn and potatoes in growth efficiency and environmental adaptability, thriving in saline or wastewater without competing for arable land [45,46]. Specific strains (Chlorella vulgaris, Tetradesmus obliquus—formerly Scenedesmus obliquus) accumulate up to 60% starch (DW) under stress [44,45,47]. Numerous studies name the Desmodesmus genus as extra tolerant for high CO₂ concentrations [18] and for wastewater treatment [24,25], which is highly relevant to solving environmental problems. Carbohydrates produced by D. armatus ARC-06 and Chlorella-group strains in commercial amounts could be used for the production of antioxidants [14], bioplastics [43], food ingredients, carrier materials [48] or as a substrate for ethanol- or methane-producing bacteria [49].

A key advantage lies in microalgal biochemical plasticity—simple cultivation adjustments can tailor biomass composition without process overhauls, enabling flexible, cost-effective production of diverse bioproducts from the same strain.

5. Conclusions

Our study primarily focused on evaluating the scalability potential of the two new promising green microalgae strains, Neochlorella semenenkoi IPPAS C-1210 and Desmodesmus armatus ARC-06, rather than conducting direct comparative analyses. Significantly, this work provides the first documented evidence of successful scalable cultivation for both C-1210 and ARC-06 strains, establishing their strong suitability for industrial-scale applications.

Three green microalgae strains—Chlorella sorokiniana IPPAS C-1, Neochlorella semenenkoi IPPAS C-1210, and Desmodesmus armatus ARC-06—demonstrated distinct protein-starch composition patterns during batch cultivation in flat-panel photobioreactors. While C-1 and C-1210 were grown under identical conditions, D. armatus ARC-06 required different growth parameters, including temperature, illumination, and medium composition. This experimental design facilitates optimization of cultivation parameters for targeted product accumulation in each strain.

Growth performance after 8 days revealed that C-1210 achieved both a high specific growth rate (2.32 ± 0.36 d⁻¹) and biomass concentration (6.76 ± 0.84 g DW L⁻¹), comparable to C-1 (2.23 ± 0.05 d⁻¹; 7.10 ± 0.41 g DW L⁻¹). D. armatus ARC-06 showed lower biomass yield (4.96 ± 0.30 g DW L⁻¹), approximately 25% less than the other strains. The maximum CUE levels for the studied strains were ranked as follows: 34% for C-1, 27% for C-1210, and 16% for ARC-06. The reduced CUE values observed in C-1210 and ARC-06 compared to the previously cultivation-optimized C-1 strain suggest the need for further refinement and optimization of cultivation conditions for these strains.

Biochemical analysis demonstrated an inverse relationship between starch and protein content: C-1 showed balanced composition (22.2% protein, 22.5% starch DW) while C-1210 accumulated higher starch (29.5% DW) and D. armatus ARC-06 exhibited exceptional starch content (51.7% DW).

These results confirm the industrial potential of all three strains, with composition adjustable through cultivation duration and conditions. The established principles for intensive cultivation are directly applicable for scaling up production of both bulk biomass and high-value compounds in flat-panel PBR systems.