Metabolic Responses of Newly Isolated Microalgal Strains Cultured in an Open Pond Simulating Reactor Under Balanced Conditions and Nutrient Limitation

Abstract

Microalgal strains—Picochlorum costavermella VAS2.5, Picochlorum oklahomense PAT3.2B and SAG4.4, Microchloropsis gaditana VON5.3, and Nephroselmis pyriformis PAT2.7—were evaluated in an Open Pond Simulating Reactor (OPSR) under varied conditions to assess their biomass yield and high-value metabolite production. Overall, the strains produced 269.1–523.0 mg/L of biomass under balanced growth conditions in modified Artificial Seawater, continuous illumination, and pH 8.5. Phosphorus limitation notably enhanced yields for SAG4.4 and PAT2.7 (529.0 ± 52.2 mg/L and 452.2 ± 21.0 mg/L, respectively). Conversely, nitrogen limitation reduced productivity. In most strains the glycolipid plus sphingolipid fraction was dominant. Significant quantities of 20:5(n-3) were traced in the cultures of VAS2.5 and VON5.3, while the PAT3.2B and SAG4.4 strains produced considerable amounts of 18:3(n-3). In contrast, the most interesting fatty acid synthesized by PAT2.7 was 16:1(n-7), which was also detected in significant quantities in VAS2.5 and VON5.3. Polysaccharide content remained stable across conditions (10–15%), and protein levels reached 45–50% under control and phosphorus-limited environments. Pigment synthesis peaked at control conditions. Overall, the biochemical profiles of these strains revealed their potential for use primarily as feed additives in the aquaculture sector.

1. Introduction

Microalgae are photosynthetic microorganisms found in all aquatic environments worldwide [1,2,3,4,5]. A key and ecologically significant characteristic of microalgae is their capacity for photosynthesis, accounting for approximately 50% of global atmospheric oxygen production. Importantly, under optimal culture conditions, microalgae can generate considerable amounts of high-value metabolites.

Genera like Picochlorum, Microchloropsis, and Nephroselmis have emerged as promising candidates for various biotechnological applications, due to their higher growth rates compared with commercial strains [6,7,8,9,10,11]. Picochlorum and Microchloropsis produce lipids with significant amounts of PUFAs, i.e., α-linolenic acid (ALA, 18:3(n-3)) and/or eicosapentaenoic acid (EPA, 20:5(n-3)) [8,11,12,13,14,15], which have been associated, among others, with the prevention of cardiovascular and neurodegenerative diseases [16,17,18]. Nephroselmis strains tend to synthesize more saturated lipids, which are more suitable for biodiesel manufacturing [8,13,19,20,21]. Nonetheless, such microalgae are considered to be important producers of proteins, providing essential amino acids to fish when used as aquafeeds, either directly or indirectly (through zooplankton feeding), and sugars [13,21,22]. Also, their pigment content, particularly carotenoids like lutein, beta-carotene, and zeaxanthin, is of high interest to various industrial sectors, e.g., food, feed, paints, etc. [6,14,23,24].

Regardless, the physiology and yields of microalgae depend on a plethora of abiotic and biotic parameters, such as the availability of nutrients (e.g., CO₂, N, P, and trace elements), pH, light intensity, and photoperiod [25,26,27,28]. For example, the abundance of N, P, and minerals is critical for biomass production, whereas nutrient limitation can enhance lipid biosynthesis. Light availability and duration are also critical parameters, with insufficient intensity or photoperiod acting as limiting factors for microalgal growth. Additionally, pH is a very important parameter, since the uptake of nutrients (including CO₂, N, and P) is heavily related to it. However, it must be highlighted that optimal conditions are species- or even strain-dependent.

The aim of the present study was the biochemical characterization of recently isolated [20] microalgal strains, i.e., Picochlorum costavermella VAS2.5, Picochlorum oklahomense SAG4.4, Picochlorum oklahomense PAT3.2B, Microchloropsis gaditana VON5.3, and Nephroselmis pyriformis PAT2.7, when cultured in an Open Pond Simulating Reactor (OPSR) containing a balanced growth medium (i.e., modified Artificial Seawater) or nitrogen- or phosphorus-limited media. An OPSR is a cost-effective and scalable type of photobioreactor which mimics large-scale photobioreactors that are often used for microalgal culture and offers the possibility to monitor and control various environmental variables, including light intensity, pH, and water temperature. The selection of the above microalgal strains was based on their interesting biochemical profiles, as showcased in previous studies [8,13,20], and due to the limited literature, especially for Picochlorum and Nephroselmis, regarding their physiology and their potential use in various biotechnological applications. Moreover, since these strains are indigenous, thus well adapted to the Mediterranean climate, they are expected to be cultured more efficiently and cost-effectively under such environmental conditions, challenging commercial strains. The selection of culture conditions was based on the goal of comparing the physiology of the selected microalgal strains under (a) balanced growth and (b) growth-limiting conditions, which presumably favor reserve material accumulation.

2. Materials and Methods

2.1. Biological Material

The green microalgal strains Picochlorum costavermella VAS2.5, Picochlorum oklahomense PAT3.2B, Picochlorum oklahomense SAG4.4, Microchloropsis (formerly known as Nannochloropsis) gaditana VON5.3, and Nephroselmis pyriformis PAT2.7 were used as biological material. These strains were previously collected from coastal areas of the Ionian Sea of Greece, treated in the laboratory until mono-algal but non-axenic using strain isolation, and identified by the use of molecular techniques, specifically by the PCR amplification of the 18S rRNA and ITS (internal transcribed spacer) gene markers [20]. Both the strain isolation process and molecular characterization are described in detail in Dritsas et al. (2023) [20].

2.2. Culture Conditions

The microalgal strains were cultured in an 8.7 L (V_w = 5 L) Open Pond Simulating Reactor (OPSR) (Figure 1). The OPSR was used for cultures under the different examined environmental conditions as a convenient and affordable type of bioreactor for lab-scale use. In detail, the dimensions of the OPSR were 29 cm × 20 cm × 15 cm (length × width × height). Transparent glass of 5 mm thickness was placed on the surface of the pond to minimize the evaporation of the growth medium and to prevent possible invasion by predators or suspended particles.

Pre-cultures were performed at a scaled level, initially from 0.25 L Erlenmeyer flasks (V_w = 0.1 L) to 1 L Erlenmeyer flasks (V_w = 0.5 L) prior to the inoculation of the cultures at 5 L with 0.2–0.5 L of microalgal pre-culture. The initial culture concentration was 1.5·10⁶ cells/mL. The basal growth medium was modified Artificial Seawater (mASW) (

Table 1. The composition of the modified Artificial Seawater (mASW) used as growth medium for control cultures and as basal growth medium for the rest of the cultures carried out in this study.

Compound	Supplier	Concentration (g/L)
NaCl	PENTA (Prague, Czech Republic)	27.0
MgSO4·7H2O	PanReac AppliChem (Darmstadt, Germany)	6.6
CaCl2	PENTA	1.5
KNO3	Scharlau (Barcelona, Spain)	1.0
KH2PO4	Himedia (Mumbai, India)	0.07
FeCl3·6H2O	BDH (Poole, UK)	0.014
Na2EDTA	Merck (Darmstadt, Germany)	0.019
Microelement solution
Compound	Supplier	Concentration (mg/L)
ZnSO4·7H2O	Merck	40.0
H3BO3	Fluka (Steinheim, Germany)	600.0
CoCl2·6H2O	Sigma-Aldrich (St. Louis, MO, USA)	1.5
CuSO4·5H2O	BDH	40.0
MnCl2	Sigma-Aldrich	400.0
(NH4)6MO7O24·4H2O	Sigma-Aldrich	370.0

), which was used for the maintenance of microalgae, as well as growth medium in the control experiments, i.e., balanced growth conditions. The growth medium was sterilized in an autoclave (Raypa AES-75, Madrid, Spain) at 121 °C for 20 min under 1.1 atm. It is noted that prior to the addition of the growth medium (4.5–4.8 L) to the OPSR, the disinfection of the pond with 70% ethanol (vol/vol) occurred.

As depicted in Figure 1, three Τ5, 8 W, 6500 Κ fluorescent lamps placed above the surface of the culture provided a continuous illumination of 232 μE/m²·s. Preliminary experiments were also carried out under the same light intensity, but with the application of a 16:8 (h, light/dark) photoperiod (see Supplementary Material). Except for the practical restrictions of the used set-ups, the selection of the aforementioned light intensity was based on the fact that most green microalgae thrive within the range of 50–400 μE/m²·s for balanced growth and photosynthetic activity.

Stirring was carried out using an Astro Liquid Filter 300 (Ace Story Aquatic, Butterworth Penang, Malaysia) water circulator with a flow rate of 150 L/h, from the inlet of which natural, moisture-saturated air was supplied to the culture at a rate of 0.5 vvm corresponding to an air flow rate of 150 L/h. Cultures were carried out in a room with a temperature of 25 ± 1 °C. During the experiments, temperature and pH values were monitored using an electronic thermometer and a pH electrode (EYELA—Model FC-10, Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and adjusted accordingly, with the addition of NaOH 2.5 M or HCl 2.5 M to 8.5 ± 0.3 or 7.5 ± 0.3, in the case of the respective cultures that were carried out in preliminary experiments (see Supplementary Material), when needed. Samples were harvested on a daily basis to estimate population growth by cell number enumeration, and at selected time points (approximately at 240 h and 450 h of culture), culture volumes of 1.5 L were harvested to determine dry biomass and various metabolic products.

The strains were cultured under balanced conditions (i.e., in mASW, having a ratio of N:P = 19:1, at pH 8.5, with continuous illumination). In addition, these strains were subjected to nitrogen- or phosphorus-limited conditions. For nitrogen-limited conditions, KNO₃ was added in concentrations of 0.1 g/L, employed for the strains of the genera Picochlorum and Nephroselmis, or 0.4 g/L, employed only for M. gaditana VON5.3. The above means that N:P was equal to ~2:1 and ~8:1 when KNO₃ was 0.1 g/L and 0.4 g/L, respectively; thus both growth media were considered as being strongly nitrogen-limited, taking into consideration that according to Redfield, a N:P ratio in the range of 16:1 offers balanced growth conditions for microalgae. Despite the lack of data in the literature regarding the ideal N:P ratio for enhanced lipid accumulation in Picochlorum and Nephroselmis, it was taken into consideration the knowledge that N:P in the range of 2:1 to 8:1 supports high lipid accumulation. Additionally, following the findings in a previous study [13] regarding NO₃⁻-N (%) uptake when these strains were cultured in an Open Pond in mASW for 19 days, the conditions ranged from 14.8 to 29.9% of the initial KNO₃ = 1 g/L. On the other hand, regarding M. gaditana VON5.3, the design of the culture in the selected N:P ratio was in accordance with evidence from the literature which presents efficacy in enhancing lipid accumulation. For phosphorus-limited treatments, KH₂PO₄ was added at 0.02 g/L instead of 0.07 g/L, i.e., a ratio of N:P of 67:1, establishing a strongly limited growth medium in phosphorus, as ratios in the range 40–80:1 are often used to study lipid accumulation or stress physiology. Moreover, in the previously mentioned study [13] the PO₄³⁻ (%) uptake of the strains used herein ranged from 60.4 to 80% of the initial KH₂PO₄ = 0.07 g/L. All other parameters remained consistent with the control set-up.

2.3. Cell Growth and Biomass Determination

The growth of microalgae was determined by the use of a Neubauer improved cell counting chamber (Poly-Optik, Bad Blankenburg, Germany) on a daily basis. This method was preferred as it offers cell count visually, thus avoiding interference from other materials, e.g., salts, cell debris, etc., which would risk the accuracy of optical density measurements. The cell counts were expressed as microalgal cell density (cells/mL) and the integrated version of Verhulst’s model (i.e., Sigmoidal Logistic function) (1) was employed as follows to estimate the parameters of growth:

N(t) = N_max/1 + b·e^1/μ·t

(1)

where N denotes the cell concentration (×10⁶ cells/mL) at time t, b is a positive constant defined as (N_max − N₀)/N₀, with N₀ representing the initial cell concentration, μ is the maximum specific growth rate (1/d), and N_max is the carrying capacity of the system. Parameter values estimation was performed by fitting Equation (1) to the experimental data using the Levenberg–Marquardt optimization method. Model fit quality was evaluated by minimizing the residual root mean square error between the experimental and model-predicted data, quantified by the values of the coefficient of determination R², which was used as a criterion for parameter optimization.

Biomass was quantified gravimetrically at designated culture time points: approximately 240 h and 450 h. Microalgal cells were harvested by centrifugation (NÜVE NF 800R, Ankara, Turkey) at 7455× g for 10 min at 4 °C, washed twice with deionized water, and dried at 80 °C until a constant weight was achieved. Biomass values were expressed as g/L.

2.4. Lipid Extraction and Purification

Total microalgal lipids were extracted using a 2:1 (vol/vol) mixture of chloroform (PENTA)/methanol (Fisher Chemical, Hampton, VA, USA), in accordance with a modified version of the Folch et al. (1957) [29] method, as adapted by Dourou et al. (2018) [30]. The purification of the obtained lipids from non-lipid components occurred as mentioned elsewhere [8]. The final solvent was evaporated under vacuum (Rotavapor R-210 evaporator—BUCHI, Flawil, Switzerland), allowing the gravimetric determination of total cellular lipids, expressed as a percentage of dry biomass (L/x%, wt/wt).

2.5. Lipid Fractionation

Microalgal lipids (~100 mg) were dissolved in 1 mL of chloroform and subjected to fractionation. The column used was of dimensions 25 × 100 mm, packed with 1 g of silicic acid (Fluka). Previously, silicic acid had been activated by heating at 80 °C overnight. Sequential elution with different solvents occurred for obtaining the fractions of neutral lipids (N), glycolipids and sphingolipids (G + S), and phospholipids (P), as described in detail elsewhere [31]. After solvent evaporation under vacuum, the lipid fractions were quantified gravimetrically and expressed as a percentage of the total lipids.

2.6. Fatty Acid Composition of Cellular Lipids

Total lipids and their fractions were converted into fatty acid methyl esters (FAMEs) and analyzed using an Agilent 7890A Gas Chromatography (GC) system (Agilent Technologies, Shanghai, China) to determine their fatty acid composition, as described in detail in the study of Dritsas & Aggelis [8].

2.7. Polysaccharide Determination

Lipid-free biomass (x_f), in the range of 20–25 mg, was hydrolyzed with 5 mL HCl 2.5 M at 100 °C for 60 min. Upon the cooling of the sample at ambient temperature, the hydrolysate was neutralized with KOH 2.5 M and filtered through Whatman No. 1 paper to remove cell debris. The reducing sugars, expressed as glucose, were quantified using the 3,5-dinitrosalicylic acid (DNS) method [32]. Intracellular polysaccharides, i.e., both storage and structural polysaccharides, were quantified as glucose equivalents and reported as a percentage of dry biomass (S/x%, wt/wt).

2.8. Protein Determination

Cellular protein was quantified from 10 to 15 mg of x_f using the biuret assay, as albumin equivalents [31], and expressed as a percentage of dry biomass (P/x%, wt/wt).

2.9. Pigment Estimation

Approximately 0.5 g of wet biomass was solved in 10 mL of ethanol 95% (vol/vol) (Fisher Chemical), prepared with Milli Q water (Honeywell, Charlotte, NC, USA). Following this, centrifugation (Hettich Mikro 200R, Föhrenstr, Germany) of the mixture at 13,300× g for 15 min at 4 °C occurred. Subsequently, 0.5 mL of the resulting supernatant was mixed with 4.5 mL of the ethanol solution [33] and spectrophotometric analysis for Chlorophyll-a, Chlorophyll-b, and carotenoids in a 1 cm² quartz cuvette occurred. The above-mentioned ethanol solution was used as the blank.

Equations (2)–(4) were used for the pigment quantification (in µg/mL) as follows [33]:

C_a = (13.36·A₆₆₄) − (5.19·A₆₄₉)

(2)

C_b = (27.43·A₆₄₉) − (8.12·A₆₆₄)

(3)

C_x+c = ((1000·A₄₇₀) − (2.13·Ch_a) − (97.63·Ch_b))/209

(4)

where A represents absorbance and Ch_a, Ch_b, and C_(exec) stand for chlorophyll-a, chlorophyll-b, and carotenoids, respectively. Pigments, evaluated as total chlorophylls (TCh) and total carotenoids (TC), were expressed as percentage in the dry biomass (TCh/x%, wt/wt, and TC/x%, wt/wt). It is noted that, in contrast to Picochlora strains and N. pyriformis PAT2.7, M. gaditana VON5.3 contains only chlorophyll-a [34]; thus the results provided in the following section refer only to chlorophyll-a and not the sum of chlorophyll-a and chlorophyll-b.

2.10. Data Treatment and Statistical Analysis

The treatment of the experimental data from the microalgal cultures and growth kinetics was carried out with the use of the graphing and analysis software OriginPro 2021 9.8.0.200 ^®, 1991–2020 (OriginLab Corp., Northampton, MA, USA).

Additionally, all experimental sets in the Tables were performed in duplicate from two biological replicates and the results are presented as mean values ± standard deviation (SD). A two-sample t-test was applied for the statistical analysis of the values regarding biomass production and reserve materials accumulation (i.e., lipids, polysaccharides, and proteins) obtained for a culture at 240 h and 450 h of culture. Statistically significant differences in biomass production, reserve materials accumulation, and the fatty acid composition of total lipids among the various culture conditions at 240 h and 450 h of culture were analyzed using a one-way ANOVA analysis of variance. Tukey’s Honest Significant Difference (HSD) post hoc test was applied to identify pairwise differences between group means. A value of p ≤ 0.05 was considered statistically significant. It is noted that the results of the statistical analyses are presented in detail in the Supplementary Material.

3. Results

The growth curves of the five examined microalgal strains, namely P. costavermella VAS2.5, P. oklahomense PAT3.2B, P. oklahomense SAG4.4, M. gaditana VON5.3, and N. pyriformis PAT2.7, under balanced growth or nutrient-limited conditions are presented in the following paragraphs of this section.

Initially, the growth performance of the five strains was assessed under balanced growth conditions, i.e., without nitrogen nor phosphorus limitation, after their culture in mASW at pH 8.5 with a 24:0 photoperiod (h light/dark).

Considering that the optimal pH for green microalgae growth typically falls within the range of 7.0 and 8.5, depending on the species and culture conditions [9,35,36], a pH value of 7.5 was additionally tested in preliminary experiments (Figure S1).

On the other hand, even though some microalgal species show increased growth and biomass productivity under continuous illumination, a 24:0 photoperiod is not always the most suitable for green microalgae, while the application of a 16:8 photoperiod is quite favorable for the growth and physiology of many green microalgae species, including ones that belong to genera like Picochlorum and Microchloropsis, which presented sufficient growth and reserve material accumulation rates [37,38,39,40]. In addition, the potential for culturing microalgal strains efficiently under a 16:8 illumination regime is of high importance for reducing electricity costs in indoor set-ups. Moreover, it could be highly useful even for examining the possibility of further using these strains in larger-scale and outdoor set-ups. Considering the aforementioned, a 16:8 photoperiod was additionally tested in preliminary experiments (Figure S2).

According to the retrieved data (see Supplementary Material, Tables S1–S10), in general, the highest yields in terms of biomass production and lipid, polysaccharide, and protein content were recorded for the cultures in mASW at pH = 8.5 and a photoperiod of 24:0. From now on, these culture conditions will be summarized under the term “control”. Therefore, the control was selected as the base to further explore the ability of the five microalgal strains to grow and synthesize high value-added metabolites under nitrogen- (mASW.N⁻) or phosphorus- (mASW.P⁻) limiting conditions (Figure 2).

3.1. Cell Growth and Biomass Production

The growth curves of P. costavermella VAS2.5 under the control and the different nutrient-limited culture conditions are presented in Figure 2a. P. costavermella VAS2.5 grew well under control conditions, gaining a biomass of 269.1 ± 6.7 mg/L. Despite the significant differences in cell number at the end of the cultures of this strain (Figure 2a), biomass production was slightly lower in mASW.N⁻ (x = 245.6 ± 8.9 mg/L) and mASW.P⁻ (x = 201.6 ± 0.4 mg/L) (

Table 2. (a) Biomass production, reserve materials accumulation, and growth parameters, (b) fatty acid composition of total lipids (TLs) and their lipid fractions (neutral—N, glycolipids—G, and phospholipids—P) of Picochlorum costavermella VAS2.5 grown in modified Artificial Seawater (mASW) and the different variations in growth in an 8.7 L (V_w = 5 L) capacity Open Pond Simulating Reactor (OPSR).

a

Growth Medium

t (h)

Biomass (x)

Lipids (L)

Polysaccharides(S)

Proteins (P)

Pigments

GrowthParameters

x (mg/L)

L/x (%)

Lipid Fractions (%)

S/x (%)

P/x (%)

TCh/x (%)

TC/x (%)

μ (1/d)

R2

N

G + S

P

Balanced growth

Control

240

100.7 ± 12.4

22.9 ± 1.9

UND

UND

UND

12.1 ± 0.1

45.1 ± 6.4

0.4 ± 0.0

0.2 ± 0.0

0.19 ± 0.00

0.97

450

269.1 ± 6.7

16.1 ± 0.0

16.6 ± 0.3

54.9 ± 6.2

28.5 ± 6.5

15.2 ± 0.3

51.0 ± 2.5

5.1 ± 0.2

2.4 ± 0.8

Nutrient limitation

mASW.N−

240

51.5 ± 0.6

3.7 ± 0.1

UND

UND

UND

8.2 ± 0.0

15.3 ± 0.0

UND

UND

0.16 ± 0.04

0.99

450

245.6 ± 8.9

10.4 ± 0.3

12.1 ± 2.5

70.5 ± 7.7

17.4 ± 1.9

10.0 ± 0.0

24.5 ± 0.7

0.3 ± 0.0

0.3 ± 0.0

mASW.P−

240

168.3 ± 28.0

5.6 ± 0.9

UND

UND

UND

7.3 ± 0.1

74.1 ± 4.1

UND

UND

0.13 ± 0.03

0.95

450

201.6 ± 0.4

16.1 ± 0.0

16.5 ± 0.2

68.7 ± 3.1

14.9 ± 3.3

1.5 ± 0.2

35.6 ± 0.4

0.7 ± 0.0

0.3 ± 0.0

b

Growth medium

t (h)

Lipid fraction

Fatty acid composition of total lipids and their fractions (%, wt/wt)

14:0

14:1(n-5)

16:0

16:1(n-7)

17:0

18:0

18:1(n-9)

18:2(n-6)

18:3(n-3)

18:4(n-3)

20:1(n-9)

20:5(n-3)

* Others

Balanced growth

Control

240

TL

5.1 ± 1.1

3.2 ± 0.1

16.5 ± 0.8

24.0 ± 4.0

<0.1

1.5 ± 0.0

25.2 ± 5.6

4.8 ± 1.8

<0.5

ND

2.5 ± 0.6

16.4 ± 3.2

1.7 ± 0.3

450

TL

4.0 ± 1.4

3.3 ± 1.0

17.2 ± 2.4

20.0 ± 4.6

<0.1

1.5 ± 0.4

30.8 ± 11.5

6.5 ± 2.6

<0.5

ND

1.9 ± 0.3

13.2 ± 4.4

1.5 ± 0.5

N

3.8 ± 1.5

3.8 ± 1.7

25.0 ± 1.5

26.4 ± 6.5

<0.5

1.9 ± 0.7

26.3 ± 12.6

2.6 ± 1.5

0.7 ± 0.0

ND

3.5 ± 2.7

3.6 ± 1.1

2.1 ± 0.9

G

9.0 ± 0.5

4.7 ± 1.8

19.1 ± 2.3

29.1 ± 2.3

<0.5

1.1 ± 0.8

6.6 ± 0.1

1.8 ± 0.3

<0.5

ND

1.9 ± 0.1

21.8 ± 3.8

2.4 ± 0.6

P

1.3 ± 0.3

1.1 ± 0.6

16.4 ± 1.4

18.0 ± 2.9

<0.5

1.3 ± 0.2

39.4 ± 10.1

7.0 ± 0.6

0.7 ± 0.4

ND

4.5 ± 2.9

9.4 ± 2.6

1.0 ± 0.1

Nutrient limitation

mASW.N−

240

TL

4.9 ± 0.4

<0.5

22.0 ± 0.4

24.6 ± 0.5

1.0 ± 0.1

<0.1

5.3 ± 0.5

1.6 ± 0.4

12.7 ± 1.0

3.0 ± 0.3

3.9 ± 0.6

13.9 ± 0.5

9.9 ± 1.0

450

TL

7.7 ± 0.6

4.7 ± 0.6

22.4 ± 1.5

30.5 ± 1.0

0.6 ± 0.1

6.5 ± 0.3

4.9 ± 1.1

1.1 ± 0.3

ND

ND

3.1 ± 0.8

21.0 ± 0.9

8.3 ± 0.2

N

6.4 ± 1.3

16.0 ± 2.8

23.4 ± 1.0

28.4 ± 2.2

0.5 ± 0.1

1.5 ± 0.0

6.6 ± 0.9

1.2 ± 0.1

ND

ND

1.5 ± 0.4

6.4 ± 0.4

7.0 ± 0.9

G

9.5 ± 0.9

2.7 ± 1.0

21.6 ± 0.4

30.7 ± 0.1

0.6 ± 0.0

1.4 ± 0.1

3.8 ± 0.3

1.1 ± 0.0

ND

ND

3.1 ± 0.0

24.6 ± 1.8

1.5 ± 0.5

P

3.0 ± 0.3

0.5 ± 0.0

21.4 ± 0.5

28.5 ± 1.0

0.9 ± 0.2

<0.5

11.8 ± 1.7

3.9 ± 0.7

<0.5

ND

6.2 ± 0.8

20.8 ± 0.1

2.5 ± 0.3

mASW.P−

240

TL

7.2 ± 0.0

2.8 ± 0.1

24.9 ± 0.4

28.5 ± 1.6

<0.5

0.6 ± 0.2

6.6 ± 0.5

1.7 ± 0.6

0.5 ± 0.3

2.3 ± 0.0

4.6 ± 2.0

19.6 ± 2.6

1.5 ± 0.2

450

TL

7.0 ± 0.2

2.6 ± 0.2

25.7 ± 0.8

26.9 ± 1.6

<0.5

1.5 ± 0.7

8.4 ± 1.3

1.4 ± 0.2

ND

<0.5

3.9 ± 0.0

17.4 ± 3.0

5.1 ± 3.4

N

4.1 ± 0.6

21.5 ± 0.2

17.2 ± 0.4

23.2 ± 2.0

ND

0.7 ± 0.0

5.8 ± 0.6

<0.5

ND

2.7 ± 0.0

3.0 ± 0.3

9.5 ± 1.6

10.8 ± 1.4

G

8.3 ± 1.0

3.1 ± 1.2

24.6 ± 0.7

32.0 ± 0.8

ND

<0.5

4.4 ± 1.6

1.4 ± 0.4

ND

<0.5

2.9 ± 0.1

19.3 ± 3.0

3.3 ± 0.8

P

5.0 ± 2.3

2.6 ± 1.7

25.6 ± 0.3

30.4 ± 0.8

ND

<0.5

9.1 ± 3.0

2.6 ± 0.8

0.9 ± 0.1

ND

6.9 ± 1.0

16.3 ± 0.0

1.0 ± 0.1

Abbreviations: (a) x (mg/L), dry biomass; L/x (%), lipids on dry biomass; N (%), neutral lipid fraction of total lipids; G + S (%), glycolipid and sphingolipid fraction of total lipids; P (%), fraction of phospholipids on total lipids; S/x (%), intracellular polysaccharides on dry biomass; P/x (%), intracellular proteins on dry biomass; TCh/x (%), total chlorophyll (chlorophyll a and b) on dry biomass; TC/x (%), total carotenoids on dry biomass; μ (1/d), maximum specific growth rate; R², R-squared statistical measure; UND, undetermined. (b) ND: not detected. * Others: mainly 10:0, 12:0, and in some cases 18:3(n-6) Note: only glycolipids (i.e., G fraction) are mentioned, as the amide bond of sphingolipids resists methanolysis during methyl esterification.

a). Statistically significant differences in biomass production between 240 h and 450 h of culture were observed in the cases of the control (p = 0.007) and mASW.N⁻ (p = 0.002) experiments. Moreover, statistically significant differences in biomass production were recorded between the sets mASW.N⁻—mASW.P⁻ at 240 h (i.e., p = 0.037), and the set control—mASW.P⁻ (p = 0.010) and mASW.N⁻—mASW.P⁻ (p = 0.034)—at 450 h of culture.

The control conditions proved more suitable for the cell growth of P. oklahomense PAT3.2B as well (Figure 2b), though, cell growth herein was markedly higher (approximately 4–6 times) compared with the rest of the examined conditions (Figure 2b). Statistically significant differences in the biomass production of each culture between 240 h and 450 h were observed. Nonetheless, the highest biomass production, significantly different from the others, was recorded in the microalgal growth in the control medium (x = 421.1 ± 30.8 mg/L with), which was confirmed by the fact that the p values in the set control—mASW.N⁻ and control—mASW.P⁻ were 0.027 and 0.022, respectively, at 240 h of culture and 0.002 and 0.003, respectively, at 450 h. On the other hand, the final biomass produced in nutrient-limited conditions ranged from 98.0 to 125.3 mg/L (

Table 3. (a) Biomass production, reserve materials accumulation, and growth parameters, (b) fatty acid composition of total lipids (TLs) and their lipid fractions (neutral—N, glycolipids—G, and phospholipids—P) of Picochlorum oklahomense PAT3.2B grown in modified Artificial Seawater (mASW) and the different variations in growth in an 8.7 L (V_w = 5 L) capacity Open Pond Simulating Reactor (OPSR).

a

Growth Medium

t (h)

Biomass (x)

Lipids (L)

Polysaccharides(S)

Proteins (P)

Pigments

GrowthParameters

x (mg/L)

L/x (%)

Lipid Fractions (%)

S/x (%)

P/x (%)

TCh/x (%)

TC/x (%)

μ (1/d)

R2

N

G + S

P

Balanced growth

Control

240

100.0 ± 9.1

13.9 ± 1.5

UND

UND

UND

13.1 ± 0.6

58.4 ± 0.9

25.2 ± 1.7

4.7 ± 0.5

0.23 ± 0.03

0.96

450

421.1 ± 30.8

11.5 ± 0.3

34.9 ± 2.3

54.4 ± 3.9

12.9 ± 1.6

8.8 ± 0.6

49.2 ± 4.8

9.0 ± 2.8

1.5 ± 0.6

Nutrient limitation

mASW.N−

240

49.3 ± 5.3

3.8 ± 0.0

UND

UND

UND

8.2 ± 0.0

15.3 ± 0.0

2.3 ± 0.3

0.7 ± 0.1

0.30 ± 0.06

0.92

450

98.0 ± 4.6

5.1 ± 1.0

UND

UND

UND

8.9 ± 0.6

29.1 ± 3.9

5.4 ± 0.7

1.0 ± 0.0

mASW.P−

240

45.2 ± 5.4

10.4 ± 0.5

UND

UND

UND

13.0 ± 1.1

28.1 ± 4.0

10.2 ± 0.9

1.7 ± 0.1

0.26 ± 0.08

0.81

450

125.3 ± 4.1

6.0 ± 2.5

UND

UND

UND

11.5 ± 2.6

45.9 ± 6.4

15.4 ± 8.8

2.8 ± 1.5

b

Growth medium

t (h)

Lipid fraction

Fatty acid composition of total lipids and their fractions (%, wt/wt)

14:0

14:1(n-5)

16:0

16:1(n-7)

17:0

18:0

18:1(n-9)

18:2(n-6)

18:3(n-3)

18:4(n-3)

* Others

Balanced growth

Control

240

TL

4.8 ± 0.7

2.7 ± 0.5

14.3 ± 0.9

2.3 ± 0.6

2.9 ± 0.7

7.5 ± 0.3

20.9 ± 2.4

4.8 ± 0.1

22.1 ± 2.4

4.5 ± 4.0

2.7 ± 0.5

450

TL

1.7 ± 0.2

6.9 ± 0.6

17.1 ± 1.8

3.0 ± 0.3

<0.5

7.3 ± 1.4

14.7 ± 0.1

21.4 ± 0.8

23.0 ± 0.6

0.5 ± 0.0

6.9 ± 0.6

N

5.6 ± 0.3

26.7 ± 2.9

10.6 ± 1.2

2.1 ± 0.3

1.6 ± 0.3

3.1 ± 0.2

9.2 ± 2.4

18.0 ± 2.0

9.5 ± 0.2

1.4 ± 0.5

26.7 ± 2.9

G

<0.5

<0.5

18.6 ± 0.2

2.1 ± 0.1

ND

13.6 ± 0.0

14.4 ± 0.6

27.5 ± 0.0

20.3 ± 0.3

3.3 ± 0.5

<0.5

P

1.0 ± 0.3

11.4 ± 2.6

11.8 ± 3.6

3.3 ± 1.0

1.3 ± 0.1

5.8 ± 2.4

12.5 ± 0.8

37.5 ± 2.0

17.7 ± 1.1

1.9 ± 0.8

11.4 ± 2.6

Nutrient limitation

mASW.N−

240

TL

0.5 ± 0.0

6.7 ± 0.4

15.2 ± 0.4

3.8 ± 0.9

6.1 ± 1.9

10.9 ± 2.7

23.3 ± 1.9

19.1 ± 8.3

13.8 ± 0.6

ND

3.7 ± 1.0

450

TL

1.1 ± 0.1

7.9 ± 0.3

14.9 ± 0.3

3.7 ± 0.1

7.2 ± 1.1

2.6 ± 0.2

17.6 ± 3.8

23.1 ± 4.0

13.6 ± 0.4

4.6 ± 0.6

3.9 ± 1.2

mASW.P−

240

TL

5.5 ± 1.0

7.6 ± 0.5

17.4 ± 2.1

3.5 ± 0.1

3.6 ± 0.0

5.9 ± 2.5

17.3 ± 2.7

25.4 ± 4.9

13.2 ± 3.2

ND

4.6 ± 0.0

450

TL

7.7 ± 3.5

10.3 ± 1.8

8.2 ± 0.0

2.9 ± 0.2

10.2 ± 2.2

15.2 ± 1.7

22.1 ± 3.5

7.9 ± 0.0

18.3 ± 0.0

ND

2.8 ± 0.0

Abbreviations: (a) x (mg/L), dry biomass; L/x (%), lipids on dry biomass; N (%); neutral lipid fraction of total lipids; G + S (%); glycolipid and sphingolipid fraction of total lipids; P (%), fraction of phospholipids on total lipids; S/x (%), intracellular polysaccharides on dry biomass; P/x (%), intracellular proteins on dry biomass; TCh/x (%), total chlorophyll (chlorophyll a and b) on dry biomass; TC/x (%), total carotenoids on dry biomass; μ (1/d), maximum specific growth rate; R², R-squared statistical measure; UND, undetermined. (b) ND: not detected, * Others: mainly 10:0, 12:0 and in some cases 18:3(n-6) Note: only glycolipids (i.e., G fraction) are mentioned, as the amide bond of sphingolipids resists methanolysis during methyl esterification.

a). No statistically significant differences were observed in biomass production between the two nutrient-limited conditions.

The growth curves of P. oklahomense SAG4.4 under the control and nutrient-limited conditions are presented in Figure 2c. Notably, the growth data of culture mASW.N⁻ also applied to the linear model (linear correlation coefficient R² = 0.87). Regarding this strain, like PAT3.2B, statistically significant differences in the biomass production of each culture between 240 h and 450 h were observed. Intriguingly, and in contrast to what was recorded for the other two Picochlora, this strain presented its highest biomass production under phosphorus-limiting conditions, i.e., x = 529.0 ± 52.2 mg/L, which was statistically significantly higher to what was recorded for the control experiment (p = 0.030) and mASW.N⁻ (p = 0.011) after 450 h of culture. Specifically, x was 299.3 ± 13.6 mg/L in the control experiment, while nitrogen limitation negatively affected the synthesis of biomass, as 195.7 ± 14.4 mg/L was produced by the end of the culture (

Table 4. (a) Biomass production, reserve materials accumulation, and growth parameters, (b) fatty acid composition of total lipids (TLs) and their lipid fractions (neutral—N, glycolipids—G, and phospholipids—P) of Picochlorum oklahomense SAG4.4 grown in modified Artificial Seawater (mASW) and the different variations in growth in an 8.7 L (V_w = 5 L) capacity Open Pond Simulating Reactor (OPSR).

a

Growth Medium

t (h)

Biomass (x)

Lipids (L)

Polysaccharides (S)

Proteins (P)

Pigments

Growth Parameters

x (mg/L)

L/x (%)

Lipid Fractions (%)

S/x (%)

P/x (%)

TCh/x (%)

TC/x (%)

μ (1/d)

R2

N

G + S

P

Balanced growth

Control

240

90.7 ± 4.9

8.6 ± 2.0

UND

UND

UND

11.2 ± 0.8

35.0 ± 1.9

1.5 ± 0.1

0.3 ± 0.0

0.21 ± 0.06

0.87

450

299.3 ± 13.6

9.4 ± 1.5

16.9 ± 0.9

68.7 ± 0.3

15.4 ± 1.1

13.1 ± 0.7

43.8 ± 2.3

4.7 ± 1.1

1.3 ± 0.0

Nutrient limitation

mASW.N−

240

80.4 ± 5.9

2.1 ± 0.4

UND

UND

UND

11.9 ± 2.7

8.9 ± 3.5

2.0 ± 1.8

0.4 ± 0.3

0.13 ± 0.07

0.90

450

195.7 ± 14.4

1.2 ± 0.0

UND

UND

UND

10.9 ± 1.1

14.2 ± 4.0

3.9 ± 0.2

0.8 ± 0.0

mASW.P−

240

95.9 ± 12.3

7.6 ± 3.0

UND

UND

UND

13.0 ± 1.1

29.5 ± 4.2

3.3 ± 0.3

0.7 ± 0.2

0.20 ± 0.03

0.95

450

529.0 ± 52.2

5.7 ± 1.6

26.1 ± 2.5

67.4 ± 3.7

5.1 ± 0.3

10.6 ± 1.5

38.2 ± 1.4

2.6 ± 0.2

0.5 ± 0.0

b

Growth medium

t (h)

Lipid fraction

Fatty acid composition of total lipids and their fractions (%, wt/wt)

14:0

14:1(n-5)

16:0

16:1(n-7)

17:0

18:0

18:1(n-9)

18:2(n-6)

18:3(n-6)

18:3 (n-3)

18:4(n-3)

20:1(n-9)

* Others

Balanced growth

Control

240

TL

4.1 ± 1.9

2.2 ± 0.6

13.4 ± 0.1

1.7 ± 0.1

2.2 ± 0.6

4.7 ± 1.1

18.4 ± 0.7

4.7 ± 1.8

7.6 ± 1.0

19.7 ± 5.0

8.5 ± 2.4

ND

12.8 ± 1.7

450

TL

1.7 ± 0.2

6.9 ± 0.6

17.1 ± 1.8

3.0 ± 0.3

<0.5

7.3 ± 1.4

14.7 ± 0.1

21.4 ± 0.8

<0.5

23.0 ± 0.6

1.0 ± 0.0

ND

4.4 ± 0.3

N

16.4 ± 0.4

13.1 ± 0.4

14.1 ± 0.5

3.1 ± 0.1

2.1 ± 0.0

0.8 ± 0.0

11.5 ± 0.4

17.3 ± 0.8

4.3 ± 0.2

13.2 ± 1.0

0.6 ± 0.2

1.6 ± 0.3

2.6 ± 0.1

G

0.5 ± 0.1

0.6 ± 0.1

18.8 ± 0.6

2.4 ± 0.0

<0.5

11.0 ± 0.1

19.0 ± 0.5

21.0 ± 0.7

ND

24.4 ± 1.5

2.1 ± 0.0

ND

<0.5

P

0.6 ± 0.1

0.8 ± 0.2

26.5 ± 0.0

5.6 ± 0.5

0.9 ± 0.2

2.5 ± 0.3

15.9 ± 1.7

26.9 ± 0.6

ND

16.3 ± 1.1

2.8 ± 0.0

ND

1.2 ± 0.5

Nutrient limitation

mASW.N−

240

TL

3.6 ± 0.4

7.6 ± 2.2

18.3 ± 1.6

7.0 ± 1.1

7.2 ± 1.2

2.6 ± 1.7

9.0 ± 0.9

5.7 ± 0.5

ND

10.3 ± 0.0

ND

ND

33.8 ± 4.9

450

TL

2.5 ± 0.0

10.1 ± 0.1

15.2 ± 0.9

4.7 ± 0.7

7.6 ± 0.1

3.1 ± 0.1

17.0 ± 0.5

19.9 ± 0.5

ND

13.5 ± 0.5

ND

ND

6.4 ± 0.5

mASW.P−

240

TL

1.1 ± 0.6

4.8 ± 1.8

17.7 ± 2.2

3.7 ± 1.2

4.4 ± 1.1

0.7 ± 0.1

18.2 ± 1.4

21.1 ± 1.5

1.7 ± 0.0

19.3 ± 0.2

3.2 ± 0.4

ND

4.9 ± 1.3

450

TL

2.6 ± 1.1

10.1 ± 1.9

13.4 ± 6.1

2.6 ± 0.2

5.8 ± 0.3

0.6 ± 0.0

15.3 ± 2.6

21.1 ± 3.2

ND

19.7 ± 3.2

2.4 ± 1.5

ND

6.4 ± 2.5

N

2.6 ± 0.2

10.7 ± 0.1

17.7 ± 0.3

3.5 ± 0.3

5.9 ± 0.7

<0.5

14.4 ± 2.0

20.9 ± 0.4

ND

18.3 ± 0.4

<0.5

ND

5.9 ± 0.2

G

0.5 ± 0.2

1.3 ± 0.1

15.5 ± 0.2

2.7 ± 0.3

13.2 ± 0.2

<0.5

16.9 ± 0.6

23.7 ± 0.1

ND

22.1 ± 0.1

3.0 ± 0.5

ND

1.3 ± 0.3

P

<0.5

<0.5

20.4 ± 1.1

5.0 ± 0.1

1.5 ± 0.0

4.8 ± 0.2

14.3 ± 0.3

32.7 ± 1.3

ND

17.7 ± 0.7

<0.5

ND

2.5 ± 0.2

Abbreviations: (a) x (mg/L), dry biomass; L/x (%), lipids on dry biomass; N (%), neutral lipid fraction of total lipids; G + S (%), glycolipid and sphingolipid fraction of total lipids; P (%), fraction of phospholipids on total lipids; S/x (%), intracellular polysaccharides on dry biomass; P/x (%), intracellular proteins on dry biomass; TCh/x (%), total chlorophyll (chlorophyll a and b) on dry biomass; TC/x (%), total carotenoids on dry biomass; μ (1/d), maximum specific growth rate; R², R-squared statistical measure; UND, undetermined. (b) ND: not detected. * Others: mainly 10:0, 12:0. Note: only glycolipids (i.e., G fraction) are mentioned, as the amide bond of sphingolipids resists methanolysis during methyl esterification.

a).

In the experiments carried out for M. gaditana VON5.3, cell growth was mostly favored under the control conditions (Figure 2d,

Table 5. (a) Biomass production, reserve materials accumulation, and growth parameters, (b) fatty acid composition of total lipids (TLs) and their lipid fractions (neutral—N, glycolipids—G, and phospholipids—P) of Microchloropsis gaditana VON5.3 grown in modified Artificial Seawater (mASW) and the different variations in growth in an 8.7 L (V_w = 5 L) capacity Open Pond Simulating Reactor (OPSR).

a

Growth Medium

t (h)

Biomass (x)

Lipids (L)

Polysaccharides(S)

Proteins (P)

Pigments

GrowthParameters

x (mg/L)

L/x (%)

Lipid Fractions (%)

S/x (%)

P/x (%)

TCh/x (%)

TC/x (%)

μ (1/d)

R2

N

G + S

P

Balanced growth

Control

240

109.3 ± 36.5

10.7 ± 2.7

UND

UND

UND

7.0 ± 0.4

18.7 ± 0.5

3.2 ± 0.8

1.5 ± 0.2

0.25 ± 0.05

0.95

450

523.0 ± 136.0

11.8 ± 3.5

34.4 ± 9.1

56.9 ± 5.7

5.4 ± 0.1

9.7 ± 0.4

21.9 ± 4.9

0.4 ± 0.1

0.6 ± 0.4

Nutrient limitation

mASW.N−

240

97.4 ± 24.6

10.1 ± 0.8

UND

UND

UND

8.7 ± 1.1

12.5 ± 1.2

3.3 ± 0.2

1.3 ± 0.2

0.24 ± 0.04

0.94

450

393.9 ± 22.1

11.9 ± 0.3

15.2 ± 4.2

71.5 ± 4.0

13.4 ± 0.2

10.7 ± 0.2

16.2 ± 2.0

2.8 ± 1.8

1.3 ± 0.3

mASW.P−

240

220.7 ± 1.0

10.2 ± 2.6

UND

UND

UND

7.3 ± 0.2

17.1 ± 4.5

0.8 ± 0.1

0.8 ± 0.1

0.20 ± 0.04

0.94

450

392.2 ± 38.0

19.2 ± 2.2

27.2 ± 9.3

55.2 ± 8.6

17.6 ± 0.6

9.7 ± 0.4

35.1 ± 3.5

0.2 ± 0.0

0.3 ± 0.1

b

Growth medium

t (h)

Lipid fraction

Fatty acid composition of total lipids and their fractions (%, wt/wt)

14:0

14:1(n-5)

16:0

16:1(n-7)

17:0

18:0

18:1(n-9)

18:2(n-6)

18:3(n-3)

18:4(n-3)

20:1(n-9)

20:5(n-3)

* Others

Balanced growth

Control

240

TL

6.9 ± 1.2

1.9 ± 1.0

23.2 ± 0.0

28.5 ± 3.6

<0.5

0.8 ± 0.4

7.5 ± 0.2

1.9 ± 0.6

1.8 ± 0.8

<0.5

3.5 ± 1.0

17.6 ± 1.4

6.2 ± 3.4

450

TL

6.6 ± 0.9

3.8 ± 0.9

22.8 ± 0.4

31.3 ± 0.8

<0.5

<0.5

6.8 ± 0.9

2.0 ± 0.5

0.7 ± 0.2

<0.5

2.9 ± 0.4

20.0 ± 1.0

2.5 ± 0.3

N

5.7 ± 1.4

3.0 ± 1.2

28.6 ± 0.4

34.9 ± 2.7

0.5 ± 0.3

0.6 ± 0.1

8.1 ± 1.2

1.4 ± 0.1

ND

1.8 ± 0.6

2.5 ± 0.6

8.5 ± 1.0

4.9 ± 0.3

G

9.1 ± 0.0

5.6 ± 0.8

20.8 ± 2.0

28.1 ± 2.8

<0.5

ND

6.4 ± 1.1

1.3 ± 0.4

ND

ND

2.0 ± 0.7

22.3 ± 3.0

4.1 ± 0.5

P

2.4 ± 0.1

0.6 ± 0.0

21.9 ± 0.2

31.8 ± 0.8

0.8 ± 0.3

<0.5

16.3 ± 1.5

4.4 ± 0.1

0.6 ± 0.2

ND

3.5 ± 0.9

15.7 ± 0.7

2.0 ± 0.4

Nutrient limitation

mASW.N−

240

TL

7.1 ± 0.0

3.6 ± 0.1

21.5 ± 3.3

28.7 ± 2.1

<0.5

<0.5

5.8 ± 0.1

2.2 ± 0.7

ND

ND

2.8 ± 0.0

20.9 ± 3.7

8.3 ± 1.4

450

TL

7.6 ± 0.5

3.7 ± 0.0

23.0 ± 1.8

26.9 ± 0.3

<0.5

<0.5

10.0 ± 4.1

1.4 ± 0.2

<0.5

ND

2.9 ± 0.1

20.8 ± 3.7

3.2 ± 1.8

N

8.3 ± 0.6

7.0 ± 0.5

26.2 ± 0.5

32.2 ± 0.6

0.5 ± 0.3

3.6 ± 2.8

7.2 ± 0.8

2.0 ± 0.6

ND

ND

2.8 ± 0.0

9.2 ± 1.3

1.1 ± 0.4

G

10.1 ± 0.2

4.1 ± 0.2

22.8 ± 0.1

30.0 ± 0.9

0.6 ± 0.3

0.6 ± 0.0

4.3 ± 0.8

1.6 ± 0.3

ND

ND

2.2 ± 0.0

22.6 ± 0.1

3.8 ± 0.7

P

3.2 ± 0.0

1.1 ± 0.1

23.7 ± 0.8

30.8 ± 0.1

0.5 ± 0.1

<0.5

15.9 ± 1.2

3.5 ± 0.2

0.6 ± 0.0

ND

4.6 ± 1.1

15.1 ± 1.7

0.8 ± 0.3

mASW.P−

240

TL

6.8 ± 1.5

3.6 ± 0.2

21.8 ± 1.3

28.7 ± 0.0

<0.5

<0.5

7.3 ± 4.0

2.7 ± 0.8

<0.5

0.9 ± 0.1

2.8 ± 0.6

20.6 ± 1.4

3.8 ± 1.2

450

TL

6.9 ± 0.1

4.8 ± 0.2

19.9 ± 0.9

29.1 ± 0.4

<0.5

<0.5

6.4 ± 2.0

1.8 ± 0.6

0.7 ± 0.3

0.6 ± 0.0

4.3 ± 0.1

22.9 ± 2.9

2.3 ± 0.9

N

5.8 ± 0.2

4.7 ± 0.3

29.9 ± 1.2

29.4 ± 1.1

0.5 ± 0.0

1.2 ± 0.1

11.4 ± 0.8

1.7 ± 0.2

ND

2.6 ± 1.0

2.9 ± 0.3

7.9 ± 1.1

2.4 ± 0.4

G

9.0 ± 0.0

4.6 ± 1.0

19.9 ± 2.9

27.3 ± 0.4

<0.5

<0.5

7.7 ± 0.3

1.4 ± 0.4

<0.5

<0.5

3.0 ± 0.3

24.3 ± 1.8

2.3 ± 0.2

P

2.7 ± 0.1

1.0 ± 0.0

18.2 ± 0.7

25.4 ± 0.5

<0.5

0.6 ± 0.1

13.9 ± 2.1

5.6 ± 2.0

2.1 ± 0.1

<0.5

7.4 ± 1.9

22.1 ± 3.0

0.8 ± 0.2

Abbreviations: (a) x (mg/L), dry biomass; L/x (%), lipids on dry biomass; N (%), neutral lipid fraction of total lipids; G + S (%), glycolipid and sphingolipid fraction of total lipids; P (%), fraction of phospholipids on total lipids; S/x (%), intracellular polysaccharides on dry biomass; P/x (%), intracellular proteins on dry biomass; TCh/x (%), total chlorophyll (chlorophyll a and b) on dry biomass; TC/x (%), total carotenoids on dry biomass; μ (1/d), maximum specific growth rate; R², R-squared statistical measure; UND, undetermined. (b) ND: not detected. * Others: mainly 10:0, 12:0, and in some cases 18:3(n-6) Note: only glycolipids (i.e., G fraction) are mentioned, as the amide bond of sphingolipids resists methanolysis during methyl esterification.

a). However, despite the small differences in cell number at the end of the cultures, the highest biomass production, i.e., 523.0 ± 136.0 mg/L, which was obtained from the control culture, was higher, but not statistically significant, compared with the biomass produced at the end of the nutrient-limited cultures, reaching 393.9 ± 22.1 mg/L and 392.2 ± 38.0 mg/L in mASW.N⁻ and mASW.P⁻, respectively (Table 5a). Herein, statistically significant differences were observed only for the biomass produced between 240 h and 450 of the mASW.N⁻ (p = 0.012) culture and the mASW.P⁻ (p = 0.046) culture.

Lastly, regarding the growth curves of N. pyriformis PAT2.7, it was recorded that mASW.P⁻ was a condition quite favorable for cell growth, as well as mASW.N⁻ (Figure 2e). However, the tendency of the cells to form cell aggregates must be highlighted as this posed a risk for the misestimation of growth. In this context, the cell count for the culture in mASW.N⁻ ceased after the 14th day of culture (i.e., at approximately 330 h) due to the increased tendency to form cell aggregates. Regarding biomass production, the highest yield was recorded when N. pyriformis PAT2.7 was cultured under the control conditions (x = 471.4 ± 27.6 mg/L) and was practically at the same level in the phosphorus-limited culture (x = 452.2 ± 21.0 mg/L) (

Table 6. (a) Biomass production, reserve materials accumulation, and growth parameters, (b) fatty acid composition of total lipids (TLs) and their lipid fractions (neutral—N, glycolipids—G, and phospholipids—P) of Nephroselmis pyriformis PAT2.7 grown in modified Artificial Seawater (mASW) and the different variations in growth in an 8.7 L (V_w = 5 L) capacity Open Pond Simulating Reactor (OPSR).

a

Growth Medium

t (h)

Biomass (x)

Lipids (L)

Polysaccharides(S)

Proteins (P)

Pigments

GrowthParameters

x (mg/L)

L/x (%)

Lipid Fractions (%)

S/x (%)

P/x (%)

TCh/x (%)

TC/x (%)

μ (1/d)

R2

N

G + S

P

Balanced growth

Control

240

137.9 ± 32.8

6.7 ± 0.2

UND

UND

UND

11.6 ± 1.1

21.8 ± 1.8

1.4 ± 0.1

0.4 ± 0.0

0.22 ± 0.03

0.95

450

471.4 ± 27.6

5.5 ± 1.6

41.5 ± 1.5

53.6 ± 3.1

5.0 ± 1.8

15.3 ± 0.9

37.8 ± 3.8

0.8 ± 0.1

0.2 ± 0.0

Nutrient limitation

mASW.N−

240

122.1 ± 15.4

3.6 ± 0.1

UND

UND

UND

11.9 ± 0.6

25.2 ± 2.2

3.5 ± 1.1

0.8 ± 0.3

** 0.24 ± 0.07

0.94

450

162.0 ± 53.0

6.2 ± 1.6

UND

UND

UND

31.6 ± 2.4

23.1 ± 3.7

1.5 ± 0.5

0.5 ± 0.2

mASW.P−

240

386.6 ± 7.6

2.5 ± 0.0

UND

UND

UND

16.2 ± 0.0

12.0 ± 0.0

0.9 ± 0.1

0.2 ± 0.0

0.25 ± 0.06

0.92

450

452.2 ± 21.0

1.7 ± 0.1

UND

UND

UND

21.8 ± 0.7

22.1 ± 0.3

2.3 ± 0.0

0.0 ± 0.0

b

Growth medium

t (h)

Lipid fraction

Fatty acid composition of total lipids and their fractions (%, wt/wt)

14:0

14:1(n-5)

16:0

16:1(n-7)

18:0

18:1(n-9)

18:2(n-6)

* Others

Balanced growth

Control

240

TL

30.2 ± 0.2

8.3 ± 0.6

9.8 ± 0.0

40.0 ± 4.1

2.6 ± 1.6

5.6 ± 1.7

1.9 ± 0.0

2.4 ± 0.3

450

TL

31.0 ± 0.7

4.4 ± 1.1

9.4 ± 0.9

39.0 ± 3.9

2.7 ± 2.0

5.4 ± 2.2

0.8 ± 0.3

7.3 ± 1.8

N

34.6 ± 1.2

5.7 ± 0.4

10.0 ± 0.6

41.1 ± 0.9

0.9 ± 0.0

2.3 ± 0.2

1.3 ± 0.1

7.7 ± 0.4

G

31.7 ± 0.2

4.7 ± 1.3

8.6 ± 0.8

39.4 ± 0.3

3.3 ± 2.2

3.9 ± 1.4

0.7 ± 0.4

4.1 ± 0.8

P

14.4 ± 1.8

10.0 ± 0.0

13.7 ± 1.1

29.6 ± 0.4

4.7 ± 0.9

17.1 ± 2.4

5.2 ± 2.8

5.8 ± 0.3

Nutrient limitation

mASW.N−

240

TL

32.2 ± 1.3

4.5 ± 0.3

11.6 ± 0.4

38.6 ± 3.2

1.3 ± 0.3

2.1 ± 0.3

ND

9.7 ± 3.2

450

TL

28.4 ± 3.8

3.4 ± 1.1

13.2 ± 1.6

42.1 ± 0.9

2.4 ± 1.1

4.5 ± 2.1

<0.1

9.7 ± 3.2

mASW.P−

240

TL

26.8 ± 0.3

3.5 ± 0.2

11.0 ± 0.1

34.9 ± 0.4

2.3 ± 0.7

9.1 ± 0.5

2.8 ± 1.1

9.7 ± 0.1

450

TL

23.3 ± 0.8

7.5 ± 0.3

9.9 ± 0.2

40.4 ± 1.3

1.7 ± 0.6

7.4 ± 2.4

1.1 ± 0.0

9.4 ± 0.1

Abbreviations: (a) x (mg/L), dry biomass; L/x (%), lipids on dry biomass; N (%), neutral lipid fraction of total lipids; G + S (%), glycolipid and sphingolipid fraction of total lipids; P (%), fraction of phospholipids on total lipids; S/x (%), intracellular polysaccharides on dry biomass; P/x (%), intracellular proteins on dry biomass; TCh/x (%), total chlorophyll (chlorophyll a and b) on dry biomass; TC/x (%), total carotenoids on dry biomass; μ (1/d), maximum specific growth rate; R², R-squared statistical measure; UND, undetermined. (b) ND: not detected. * Others: mainly 10:0, 12:0. Note: only glycolipids (i.e., G fraction) are mentioned, as the amide bond of sphingolipids resists methanolysis during methyl esterification. ** Cell count ceased at approximately 330 h of culture due to the increased tendency of the cells to form cell aggregates.

a). However, the biomass produced under nitrogen-limited conditions was at significantly lower levels at 162.0 ± 53.0 mg/L (Table 6a). Specifically, p was 0.019 in the set control mASW.N⁻ and in the set mASW.N⁻—mASW.P⁻, with 0.006 and 0.023 at 240 h and 450 h, respectively. Notably, statistically significant differences in the biomass production of the same culture were recorded only under control conditions (p = 0.016).

3.2. Synthesis of Storage Materials and Fatty Acid Composition of Total Lipids and Their Fractions

P. costavermella VAS2.5 showed an adequate to relatively high lipid reserve accumulation capacity under nutrient-limited conditions. To be more specific, the cultures performed in control conditions and under phosphorus limitation led to L/x% ≈ 16% (wt/wt) at the end of both cultures (Table 2a). A statistically significantly lower lipid content, compared with the above (p = 0.0004) was recorded for this strain under nitrogen-limitated conditions (L/x% = 10.4 ± 0.3%, wt/wt). The major lipid fraction in all culture conditions was that of G + S, in high percentages (i.e., 54.9–70.5%, wt/wt), while lipid fractions N and P appeared in lower and comparable proportions (Table 2a). Regarding the polysaccharide content of the biomass, percentages in the range of S/x% = 10–15.2 (wt/wt) were observed between the culture in control conditions and mASW.N⁻, which were statistically significantly different (p was 0.0001 and 0.0008, respectively, at 240 h and 450 h of culture). The polysaccharide content of the culture in mASW.P⁻, which at the end of the culture was S/x% = 1.5 ± 0.2%, wt/wt, was, as expected, significantly lower than the other culture conditions at both time checkpoints as well. Also, the protein content was high, especially in the control medium where it exceeded 50%, wt/wt, while in the other culture conditions the percentage ranged between 24.5 and 35.6%, wt/wt (Table 2a). Statistically significant differences were recorded in all cases herein, independently of the time of incubation or culture condition. Finally, total chlorophyll and carotenoids values were lower in mASW.N⁻ and mASW.P⁻ compared with the control medium (Table 2a).

The composition of total lipids and their lipid fractions in fatty acids of P. costavermella VAS2.5 when cultured in control and nutrient-limited conditions is shown in Table 2b. The main PUFA synthesized under all culture conditions was 20:5(n-3), ranging from 13.2 to 21.0% (wt/wt) with the most favorable condition being nitrogen limitation. Notably, the content of 20:5(n-3) was slightly higher, i.e., 23.9 ± 0.3% (wt/wt), when this strain was cultured in conditions similar to the control but at pH = 7.5 (Table S1), while it was lower, i.e., 8.0 ± 1.0% (wt/wt), when a 16:8 photoperiod was applied (Table S2). Similar percentages of 20:5(n-3) were also present in the polar lipid fractions per case (9.4–24.6%, wt/wt), while, in general, it was lower in the N lipid fraction (i.e., 3.6–9.5%, wt/wt) (Table 2b). Apart from 20:5(n-3), the other fatty acids produced in significant proportions were 16:0 (17.2–25.7%, wt/wt) and 16:1(n-7) (20–30.5%, wt/wt), with their percentages in the lipid fractions changing at similar levels to those determined in total lipids. Similar results were also obtained at cultures that were carried out at pH = 7.5 with constant illumination and at pH = 8.5 with a 16:8 photoperiod (Tables S1 and S2). It is also noted that in some cases 18:4(n-3) was detected. Regarding the composition of total lipids in fatty acids, statistically significant differences were observed for the following sets: (a) control—mASW.N⁻: 14:1(n-5), 16:0, 17:0, 18:1(n-9), 18:3(n-3), and 18:4(n-3) at 240 h and 17:0, 18:0, 18:3(n-3), and 18:4(n-3) at 450h; (b) control—mASW.P⁻: 16:0 and 18:0 at 240 h and C17:0, 18:3(n-3), and 18:4(n-3) at 450 h; (c) mASW.N⁻—mASW.P⁻: 14:1(n-5), 17:0, and 18:3(n-3) at 240 h and 18:0, 18:3(n-3), and 18:4(n-3) at 450 h.

P. oklahomense PAT3.2B presented a lower lipid content (L/x% = 5.1–6.0%, wt/wt) regardless of the growth conditions at the end of culture compared with the control experiment (L/x% = 11.5 ± 0.3%, wt/wt). At the end of culture, no statistically significant differences were observed in terms of lipid accumulation. As mentioned before, the produced lipids were predominated by G + S (54.4 ± 3.9%, wt/wt), followed by N (34.9 ± 2.3%, wt/wt) and P (12.9 ± 1.6%, wt/wt) (Table 3a). Polysaccharide content ranged between 8.8 and 11.5% (wt/wt). Notably, statistically significant differences were observed only at 240 h of culture, in the sets control—mASW.N⁻ (p = 0.026) and mASW.N⁻—mASW.P⁻ (p = 0.027). On the other hand, protein synthesis was particularly high under control conditions and phosphorus limitation (P/x% = 49.2 ± 4.8%, wt/wt and P/x% = 45.9 ± 6.4%, wt/wt, respectively). Regarding protein content, the only significant difference in terms of statistical analysis was observed at 240 h of the set control—mASW.N⁻ (p = 0.002). Lastly, a high concentration of total chlorophyll and carotenoids was recorded in all the conditions tested, especially under phosphorus-limited conditions (Table 3a).

In contrast to P. costavermella VAS2.5, the fatty acid composition of the total lipids and their fractions, where it was possible to be analyzed, of P. oklahomense PAT3.2B revealed 16:0, 18:1(n-9), 18:2(n-6), and the PUFA 18:3(n-3) (13.6–23.0%, wt/wt) as the predominant fatty acids, with similar percentages in both the G and P fractions but lower in N when the microalgae were grown under control conditions (Table 3b). Similar percentages were recorded for 18:3(n-3) synthesis when this strain was cultured in mASW at pH = 7.5 with constant illumination or in mASW at pH = 8.5 with a 16:8 photoperiod (Tables S3 and S4). Other fatty acids, at significantly lower percentages, synthesized by this strain under the examined culture conditions were 14:1(n-5), 18:0, 16:1(n-7), and 14:0. Finally, an unusual finding was the presence of 17:0 at 7.2–10.2%, wt/wt, under nitrogen- and phosphorus-limiting conditions (Table 3b), as well as at 240 h of the culture that was performed at pH = 7.5 (Table S3). Regarding the composition of total lipids in fatty acids, statistically significant differences were observed for the following sets: (a) control—mASW.N⁻: 14:1(n-5) at 240 h and 18:3(n-3) at 450 h; (b) control—mASW.P⁻: 14:0 at 240 h and 14:1(n-5), 16:0, 17:0, 18:0, and 18:4(n-3) at 450 h; (c) mASW.N⁻—mASW.P⁻: 16:0, 18:0, 18:2(n-6), and 18:3(n-3) at 450 h.

Similarly to the other P. oklahomense strain, SAG4.4 lipid synthesis was not affected positively by nutrient limitation. In fact, L/x% ranged from 1.2 to 5.7%, wt/wt, which was lower compared with the value recorded for the strain’s culture under control conditions (L/x% = 9.4 ± 1.5%, wt/wt) (Table 4a). Notably, lipid content was statistically significantly different in the set control—mASW.N⁻ at 450 h of culture (p = 0.040). The major lipid fraction in the control experiment and mASW.P⁻, when it was possible to determine the lipid fractions, was that of G + S, which obtained values ranging between 67.4 and 68.7% (wt/wt), followed by N and P (Table 4a). The biosynthesis of polysaccharides was similar between the different conditions, with their concentration ranging between 10.6 and 13.1%, wt/wt. The above was confirmed by the fact that no statistically significant differences were observed for the three culture conditions. The produced biomass was rich in proteins after culture at control conditions and in mASW.P⁻ (P/x% = 38.2–43.8%, wt/wt). Yet, clearly lower protein levels were determined in mASW.N⁻ (P/x% = 14.2 ± 4.0%, wt/wt), which were statistically significantly different to the other two cultures at both 240 h and 450 of culture (Table 4a). Finally, regarding the values of pigments, these ranged between 2.6 and 4.7%, wt/wt, for total chlorophyll and between 0.5 and 1.3%, wt/wt, for carotenoids (Table 4a).

In general, the fatty acid composition of total lipids and their lipid fractions, where possible, of the other P. oklahomense strain, SAG4.4, was similar to the one described for P. oklahomense PAT3.2B (Table 4b). Specifically, the major PUFA synthesized under all culture conditions was 18:3(n-3), the concentration of which ranged from 13.5 to 23.0% (wt/wt), with the most favorable condition for its synthesis in the control experiment. Similar to the above were the percentages of this fatty acid in the individual lipid fractions. Other fatty acids produced in significant concentrations were 18:1(n-9) (14.7–17.0%, wt/wt) and 16:0 (13.4–17.1%, wt/wt), with their percentages in the various lipid fractions being generally similar to those of total lipids. The detection of the fatty acids 18:3(n-6) and 18:4(n-3) in some cases is noted as well. Similar profiles regarding the fatty acid composition of total lipids of this strain were recorded for this strain when cultured under control conditions but at pH = 7.5 instead of pH = 8.5 or with a 16:8 photoperiod instead of 24:0 (Tables S5 and S6). Regarding the composition of total lipids in fatty acids, statistically significant differences were observed for the following sets: (a) control—mASW.N⁻: 18:1(n-9) at 240 h and 17:0, 18:0, and 18:3(n-3) at 450 h; (b) control—mASW.P⁻: 17:0 and 18:2(n-6) at 240 h and 18:0 at 450 h; (c) mASW.N⁻—mASW.P⁻: 18:2(n-6) at 240 h and 17:0, 18:1(n-9), and 18:3(n-3) at 450 h.

With regard to the ability of M. gaditana VON5.3 to accumulate reserve materials under the examined culture conditions, the highest lipid content was determined in cells when growing in mASW.P⁻, exceeding L/x% = 19% (wt/wt), followed by the cultures under nitrogen limitation and control conditions. The main lipid fraction in all the culture conditions tested, like Picochlora, was that of G + S, ranging between 55.2 and 56.9% (wt/wt), with the exception of the cultures in mASW.N⁻ in which the percentage of G + S reached 71.5 ± 4.0%, wt/wt (Table 5a). The following lipid fraction was that of N and then of P lipids (Table 5a). Regardless of the culture condition, polysaccharide content remained practically unchanged and at the level of 10% (wt/wt) in this strain, while protein accumulation seemed to be favored under phosphorus-limiting conditions, in which the concentration reached 35.1%, wt/wt, compared with 16.2–21.9%, wt/wt, in the other culture conditions (Table 5a). In this set of experiments, no statistically significant differences were recorded regarding the accumulation of reserve materials, regardless of the culture condition and the time of incubation. Finally, the pigment content was significantly higher when this strain grew in mASW.N⁻ (Table 5a).

In the case of M. gaditana VON5.3, the major PUFA synthesized, as traced in the fatty acid composition of total lipids and lipid fractions (where possible), under all culture conditions was 20:5(n-3), ranging from 20.0 to 22.9% (wt/wt), with the most favorable condition being the culture in mASW.P⁻ (Table 5b). A slightly higher content of 20:5(n-3), i.e., 24.4 ± 1.5% (wt/wt), was exhibited by this strain when cultured under similar conditions to the control experiment but at pH = 7.5 (Table S7). In general, the percentages of 20:5(n-3) in the individual lipid fractions were in the same range as those in the total lipids in polar lipid fractions, with minor variations. On the contrary, for example, the concentration of this fatty acid was significantly lower in the N fraction, regardless of the culture conditions, ranging between 7.9 and 9.2%, wt/wt. Other fatty acids produced in significant levels were 16:0 (19.9–23.0%, wt/wt) and 16:1(n-7) (26.9–31.3%, wt/wt) acids, with the percentages in their lipid fractions ranging at similar levels to those determined in total lipids. The production of these fatty acids was comparable when M. gaditana VO5.3 was cultured in mASW at pH = 7.5 and a 24:0 photoperiod or at pH = 8.5 with a 16:8 photoperiod (Tables S7 and S8). It can also be noted that 18:4(n-3) was detected in all cultures except for the cultures performed under nitrogen limitation (mASW.N⁻) (Table 5b, Tables S7 and S8). Regarding the composition of total lipids in fatty acids, statistically significant differences were observed for the following sets: (a) control—mASW.N⁻: 17:0 at 240 h and 16:1(n-7) at 450 h; (b) control—mASW.P⁻: 17:0 and 18:4(n-3) at 240 h and 18:4(n-3) at 450 h; (c) mASW.N⁻—mASW.P⁻: 18:4(n-3) at both 240 h and 450 h.

The accumulation of lipid reserves in the case of N. pyriformis PAT2.7 was even lower, regardless of the culture conditions, with L/x% ranging from 1.7 to 6.2%, wt/wt. Notably, statistically significant differences were observed among the three examined conditions at 240 h. As previously mentioned, the predominant lipid fraction was G + S in the culture performed under control conditions, followed by N and P lipid fractions (Table 6a). Regarding the polysaccharide content of the biomass, a high accumulation rate was observed under nitrogen- (S/x% = 31.6 ± 2.4%, wt/wt) and phosphorus- (S/x% = 21.8 ± 0.7%, wt/wt) limitated conditions at the end of the cultures (Table 6a). The statistical analysis revealed that the polysaccharide content was significantly higher at 240 h of culture in mASW.P⁻, i.e., p was 0.040 and 0.048 for the sets control—mASW.P⁻ and mASW.N⁻—mASW.P⁻, respectively. Similarly, at 450 h, the statistical analysis confirmed what was recorded for mASW.N⁻. Moreover, the protein content was significant in all the culture conditions tested (P/x% = 22.0—37.8%, wt/wt) and especially under control conditions (P/x% = 37.8% ± 3.8%, wt/wt) (Table 6a). The protein content was statistically significantly lower compared with the other two culture conditions, i.e., p was 0.049 and 0.022, respectively, for the sets control—mASW.P⁻ and mASW.N⁻—mASW.P⁻. Finally, regarding the values of pigments, there was a variation of 0.8–2.3%, wt/wt for total chlorophyll, while carotenoids were detected at lower concentrations, up to 0.5%, wt/wt (Table 6a).

Lastly, the composition of total lipids and lipid fractions (where possible) in fatty acids of N. pyriformis PAT2.7 when cultured under control and nutrient-limited conditions is shown in Table 6b. The predominant fatty acids were 16:1(n-7) (39–42.1%, wt/wt) and 14:0 (23.3–31%, wt/wt). It is noted that the synthesis of both fatty acids was comparable to the above when this strain was cultured in mASW at pH = 7.5 with constant illumination but lower when cultured in mASW at pH = 8.5 and a 16:8 photoperiod (Tables S9 and S10). The respective contents of the N and G lipid fractions with respect to these fatty acids were similar to those of TLs. However, for the P lipid fraction, a generally reduced presence of both of the above fatty acids was recorded, compared with TLs, and the two aforementioned lipid fractions. Regarding the composition of total lipids in fatty acids, statistically significant differences were observed for the following sets: (a) control—mASW.N⁻: 14:1(n-5) and 16:0 at 240 h; (b) control—mASW.P⁻: 14:1(n-5) at 240 h; (c) mASW.N⁻—mASW.P⁻: 14:0 and 18:1(n-9) at 240 h.

4. Discussion

Microalgae are used in a variety of biotechnological applications, while, at the same time, they are an important biological material for basic research. All strains used in the current study were previously characterized biochemically, exhibiting profiles of interest, and cultured under laboratory conditions [8,13,20]. In the following paragraphs, a comparison and discussion of the results of this study with previous studies will occur, in order to expand the discussion regarding the optimum culture conditions for these strains, as well as giving prominence to the strains’ plasticity of growing in different illumination intensities, temperatures, aeration, agitation, etc.

In general, and taking into account the relevant data in the literature, the strains grew adequately, and, intriguingly, in some cases outperformed other strains of the same species or even other strains of microalgae that are commercially exploited. The predominant reserve material of all microalgae was proteins, followed by polysaccharides and lipids. In some cases, lipid biosynthesis was favored under phosphorus-limiting conditions, while nitrogen limitation seemed to inhibit the biosynthesis of biomass and reserve materials. In the same context, cultures in mASW under constant illumination at pH = 7.5 led to enhanced lipid synthesis in some cases, whereas the application of a 16:8 photoperiod when the strains were cultured in mASW at pH = 8.5 negatively affected the biosynthesis of biomass and reserve materials (see Supplementary Material). In the following paragraphs, a detailed discussion about the findings of this research and a comparison with the literature occurs.

4.1. Cell Growth and Biomass Production

Initially, regarding the three Picochlorum strains, in addition to the growth that occurred under both nutrient-limitated conditions, all strains grew despite the culture conditions. However, in most of the cases, there was less biomass compared with their cultures under control conditions in the OPSR, Erlenmeyer flasks, or Stirred Tank Reactor (STR), with cultures that lasted approximately 450 h and 250 h, as presented elsewhere [8,20]. The above seems to confirm that for these strains the light supply played a crucial role in growth. In the cultures that were carried out in the STR and Erlenmeyer flasks it was possible to provide high light intensity (i.e., 1071 μE/m²·s and 387 μE/m²·s, respectively). On the other hand, de la Vega et al. (2011) [7] cultured Picochlorum sp. HM1 at luminous intensities of 100 and 1200 μE/m²·s and observed a slight elevation in specific growth rate (from 0.031 1/h to 0.034 1/h), suggesting that the growth of this strain approaches saturation at relatively low light intensities, while no evidence for photoinhibition was observed at elevated light intensities. However, the light energy supply of the cells can be severely affected by so-called “mutual shading”, a condition where denser populations block light from penetrating deeper into the microalgal culture as cell concentration increases [41,42,43]. In the OPSR, light energy was not only of relatively low light intensity, but also introduced from the top of the bioreactor. Consequently, as cell density grew, light penetration diminished at the lower layers, creating suboptimal illumination that adversely impacted the metabolism of cells to an extent.

It is noteworthy that the culture of P. oklahomense SAG4.4 in mASW.P⁻ led to almost double biomass production compared with the control conditions, indicating the intra-species variability regarding the response under similar environmental conditions, as documented for these strains in Dritsas et al. (2023) [20] as well. The importance of phosphorus for the growth of Picochlorum has also been studied by other researchers. For example, in another study, cultures of a Picochlorum sp. in 0.5 M MD4 media supplemented with varying concentrations of phosphorus in the form of KH₂PO₄, like this study, were carried out [44]. According to the findings of that study, KH₂PO₄ 0.16 g/L displayed the highest cell density from day 18 to 24, a concentration which was more than double compared with the 0.07 g/L or 0.02 g/L of mASW.P⁻ in the cultures performed herein. In another study, biomass productivity decreased by nearly 30% and 57% following Picochlorum sp. cultures in a medium supplemented with half or none NaH₂PO₄. 2H₂O compared with the control experiment [39].

On the other hand, nitrogen is a nutrient of high importance for the growth of microalgae; thus its availability is considered as one of the primary factors affecting yields. Moreover, nitrogen is one of the nutrients that is most rapidly depleted during culture under laboratory conditions. Therefore, the low yields observed for the examined Picochlora, especially for the two P. oklahomense strains, came as no surprise when cultured under nitrogen limitation (mASW.N⁻). Similar findings were reported by El-Kassas (2013) [39] after growing a Picochlorum strain under nitrogen limitation/starvation conditions, who observed a negative correlation between biomass production and the low concentration of nitrogen, administered in the form of NaNO₃. The above was in agreement with the conclusion reached by Vo et al. (2023) [45] for another strain of the genus. However, it cannot be neglected that high nitrogen concentrations seem to negatively affect growth as well [39]. According to a recent study, L-Asparagine posed as a more suitable nitrogen source for improved yields in Picochlora [46].

M. gaditana VON5.3 presented slightly lower growth rates when grown in mASW.N⁻ and mASW.P⁻ compared with the culture under control conditions. Yet, this strain produced more biomass compared with the two nutrient-limited culture conditions, in which biomass production was at similar levels, denoting the sufficiency of the initial concentration of both nutrients. Interestingly, all M. gaditana VON5.3 cultures in the OPSR led to the production of more biomass than Microchloropsis salina (formerly known as Nannochloropsis salina) (x_max ≈ 0.3 g/L) which was grown in mASW for 16 days under different light intensities [47]. Additionally, it is worth mentioning that M. gaditana VON5.3, growing under control conditions, produced almost twice the biomass compared with the M. gaditana strain used by Dourou et al. (2018) [30] under similar culture conditions. The abovementioned becomes even more interesting when considering that their culture lasted more than 100 hours longer compared with the one presented herein. Nonetheless, Dourou et al. (2018) [30] confirmed the importance of a high-concentration phosphorus supply in the growth environment of M. gaditana, as their study recorded a positive correlation between the provided phosphorus and biomass production. On the same note, recently, Kim et al. (2024) [47], culturing Μ. gaditana CCMP 526 (presented as N. gaditana CCMP 526) under N- and P-limitated conditions, recorded lower cell numbers by 36% and 11%, respectively, compared with the control conditions. Similar findings were presented by Cecchin et al. (2020) [34] under N-limited conditions. However, the supplementation of the culture medium with urea seems to be beneficial for microalgal growth [48].

The cultures of M. gaditana VON5.3 in the OPSR, regardless of the culture conditions, after 240 h of culture were significantly lower compared with the biomass production that was recorded when this strain grew in STR at 25 °C, [8], as well as compared with the cultures carried out in Erlenmeyer flasks [20]. However, biomass production was comparable with what was recorded in the other studies only in the case of the culture under control conditions after 450 h of incubation [8,20], showcasing the dependence of microalgae on light intensity. In addition, a study on M. salina showed a positive correlation of the increase in biomass production with the intensity of the provided light energy [49]. On the other hand, another M. salina grown under similar culture conditions in OPSR, but at 120 μE/m²·s, produced a biomass of equivalent amount to that of M. gaditana VON5.3 [31]. In our study, the cross-shading of the cells from the 10th day of culture might hinder the growth of the culture. However, it should also be taken into consideration that according to Simionato et al. (2011) [50], significant alterations in biomass production are evident only under extreme light intensities, whereas incubation within a defined light energy threshold causes little changes in biomass production.

Lastly, biomass production by N. pyriformis PAT2.7 was comparable under control conditions and in mASW.P⁻, whereas culture in mASW.N⁻ resulted in nearly a threefold reduction compared with the other two set-ups. Similar findings on nitrogen limitation are also reported by Mastropetros et al. (2023) [21]. Notably, cell counting in mASW.N⁻ was terminated prematurely, as from 330 h of culture a strong tendency to form aggregates of cells was observed, indicative of the stress induced.

Similarly to the above, the growth of this strain in STR at 25 °C [8] and in Erlenmeyer flasks [20] led to higher biomass production compared with what was recorded herein. This observation suggests once again the dependence of microalgae on light intensity. However, similarly to what was previously discussed, the cross-shading from the 10th day of culture played an important role since the cultures presented a dark green color, indicating the insufficient availability of the supplied light in the lower layers of the culture. Moreover, it is of interest to mention the apparent plasticity in the response to different temperature values, with the optimal growth temperature varying between species or even strains of Nephroselmis species. For instance, the ability of Nephroselmis to grow at 20–21.5 °C, marking relatively high biomass production, has been reported [8,9], while other representatives of the genus grow efficiently at higher temperatures (e.g., 25–27 °C) [19,23,51]. In any case, it is worth mentioning that the enrichment of the culture medium with vitamin B12 has been shown to positively affect the production of biomass by Nephroselmis astigmatica [52].

4.2. Synthesis of Storage Materials

The cultures of P. costavermella VAS2.5 under control conditions and phosphorus limitation favored lipid synthesis. The same was the case for the two P. oklahomense strains as well. Phosphorus limitation, as well as nitrogen limitation, though to a lesser extent, proved to be a particularly favorable condition for lipid accumulation in other Picochlorum strains as well [39,53], while Nguyen et al. (2024) [44] reported higher lipid accumulation when KH₂PO₄ = 0.16 g/L. In contrast, lipid accumulation in all Picochlora cultures used herein was negatively affected by nitrogen limitation. Undoubtedly, the above can be directly correlated with the reduced biomass production of the strains under these conditions. However, when Anto et al. (2024) [46] cultured Picochlorum sp. NITT 04 under various combinations of N and P, lipid content maximization under a N:P ratio of 1.4:0.6 and salinity of 24 ppt (i.e., L/x% ≈ 50%, wt/wt) was observed.

Similar levels of polysaccharide content were recorded under the different culture conditions for the three strains, except for the culture of P. costavermella VAS2.5 in mASW.P⁻, where intracellular polysaccharides were almost fully consumed by the end of the culture. Such physiological characteristics are strongly related to the need of the cells for energy and metabolic precursors at the expense of the reserve materials when growing under environmental stressors. In addition, in some cultures, a decrease in lipid stock was noted which was accompanied by an increase in polysaccharide concentration, and vice versa, indicating that the two biosynthetic pathways are competitive with each other, or even that the biotransformation of sugars to lipids and vice versa occurred. According to Bellou & Aggelis (2012) [31], these physiological characteristics can be attributed to the need of the cells for energy and metabolic precursors at the expense of the reserve materials when growing under environmental stressors.

High protein accumulation was also observed for all strains herein, in the range of 35–50% (wt/wt). This corroborates earlier predictions, since the ability of Picochlora to accumulate proteins at high levels has been well documented [8,11,13,14,20,39,53,54]. However, the protein content of the examined Picochlorum strains cultured in mASW.N⁻ was significantly lower compared with the other cultures, ranging from 14 to 25% (wt/wt). These findings are in accordance with what El-Kassas (2013) [39] described for other Picochlorum. Specifically, under nitrogen starvation conditions, other Picochlorum strains had shown dramatic reductions and low rates of intracellular protein accumulation when growing on various substrates and under various conditions [39]. In addition, El-Kassas et al. (2013) [39] recorded a significant decrease in protein content under phosphorus-limitated conditions, which in this case seems to be verified only in the case of P. costavermella VAS2.5, in which a decrease from 74.1 ± 4.1% at 240 h to 35.6 ± 0.4%, wt/wt at 450 h of culture was recorded. In any case, the fact that members of Picochlorum possess amino acid profiles of high commercial and biotechnological interest is of particular value. For instance, according to Dritsas et al. (2025) [13], P. costavermella VAS2.5 and P. oklahomense SAG4.4 proved to be great producers of lysine and threonine.

Finally, with regard to the pigments produced, i.e., chlorophylls and carotenoids, which usually are rich in lutein and zeaxanthin in Picochlora [7,14,55], generally similar or lower production was observed compared with other studies. Notably, in all measurements, chlorophyll-a was produced in significantly greater amounts than chlorophyll-b. This pattern aligns with the findings from previous studies on other strains within the genus [14,53]. However, nutrient limitation proved to be less suitable for the greater synthesis of pigments by Picochlorum strains in comparison with the control culture conditions. Nonetheless, the pigment content of the cells is influenced by many parameters, such as the growth phase of the culture, the intensity of the light energy supplied, and the CO₂ concentration [56]. In certain microalgae, e.g., H. pluvialis, Dunaliella salina, providing a high light intensity or gradually increasing the temperature in the culture environment, parameters that stress cells and activate the photoprotection mechanisms of microalgae, is considered to be a safe and effective strategy to increase carotenoid synthesis [57]. The aforementioned seem to apply in Picochlorum as well, some species of which can grow sufficiently under light intensities of up to 2000 μE/m²·s and temperatures up to 39 °C [6,8,58]. Another influential parameter is nutrient availability. However, the phosphorus concentration in the culture medium did not seem to affect high pigment content in the study of Nguyen et al. (2024) [44]. On the other hand, LaPanse et al. (2024) [54] highlighted the trend of Picochlorum celeri reducing pigment content under nitrogen limitation, as the cells direct cellular metabolism towards polysaccharide production and away from nitrogen-rich macromolecules. However, another important parameter that can affect chlorophyll production is the concentration of Fe in the microalgal growth medium [59]. In particular, experiments conducted on a P. oklahomense (presented as P. oklahomensis) strain presented that iron limitation significantly reduced total chlorophyll per cell, up to 41% when the initial Fe concentration was half that of the control experiment [59], and a decrease that correlated with Fe concentration was also shown for the carotenoids produced.

In contrast to what was observed for the P. oklahomense strains used in this study, the most favorable condition for lipid accumulation in M. gaditana VON5.3 proved to be phosphorus limitation, since it approached 20% (wt/wt) at the end of culture. Interestingly, the lipid content was slightly higher than that of the commercial strain of the ‘sibling’ genus Nannochloropsis, such as Nannochloropsis sp. from Reed Mariculture (Campbell, CA), which exceeded 17% (wt/wt), but was lower compared with what was recorded for Nannochloropsis oceanica F&M-M24, which approached 30% (wt/wt) [60,61]. Similar results to this study were obtained by Dourou et al. (2018) [30] who recorded 2–3 times higher lipid accumulation levels when another strain of the species was grown under phosphorus-limiting conditions, exceeding up to 30% (wt/wt). On the other hand, nitrogen limitation did not seem to favor lipid accumulation, which was at similar levels to the control experiment. In agreement with these findings are the results of another study on M. salina, which reported that lipid production was not induced under nitrogen-limitated conditions [62], whereas the opposite conclusions were reached by other studies [63,64,65]. Therefore, the determination of the proper concentration of the nitrogen source is of most importance for the optimization of lipid yields.

The polysaccharide content of M. gaditana VON5.3 under nutrient limitation was slightly increased compared with the control medium. On the other hand, the results in this case were comparable to what was recorded for the cultures of this strain under the other balanced growth conditions of cultures in Erlenmeyer flasks [20] and the STR [8]. Moreover, despite the comparable amounts of polysaccharides produced by the M. salina strain that Bellou & Aggelis (2012) [31] used, it must be pointed out that Microchloropsis strains are able to produce polysaccharides in the range of 20–25% (wt/wt) of their dry biomass [30].

Among the cultures of M. gaditana VON5.3 in the OPSR under nutrient-limited conditions, mASW.P⁻ stood out in terms of protein synthesis, at approximately 35% (wt/wt). Notably, this protein content was significantly higher than what was recorded for the cultures of this strain in Erlenmeyer flasks and the STR [8,20]. Notably, the protein content after culture in mASW.P⁻ was comparable to that of other Microchloropsis strains [11,30,31]. On the contrary, it came as no surprise that the lowest protein content was recorded under nitrogen-limitated conditions.

Finally, M. gaditana VON5.3 showed a relatively high pigment content, which was at similar levels for all tested culture conditions and in most of the cases comparable to the cultures of this strain in other types of photobioreactors [8,13,20]. This feature offered an increased capacity in light energy uptake and contributed positively to their adaptation under the given culture conditions. The sole exception was the culture in mASW.P⁻, in which the chlorophyll composition was very low. In another study, nitrogen limitation decreased photosynthetic activity and chlorophyll content, while phosphorus limitation led to an increase in carotenoid content [47]. Overall, since Microchloropsis are known producers of carotenoids containing high levels of violaxanthin and β-carotene [47,66], it is important to optimize the conditions that will enhance their production levels.

Lastly, the lipid production of N. pyriformis PAT2.7 was low, regardless of the culture conditions. Neither nitrogen nor phosphorus limitation led to enhanced lipid synthesis rates for N. pyriformis PAT2.7. Although the available literature on the ability of strains of this genus to accumulate lipids is quite limited, the results of the present study are comparable to those of another Nephroselmis strain (i.e., L/x% = 10.5%, wt/wt), which was grown for about one week in a nutrient medium at 25 °C, with a salinity of 28‰ and a light intensity of 80–100 μE/m²·s [67]. Nephroselmis sp. (var. Messolonghi) of Hotos et al. (2023) [68] grown in Walne medium accumulated lipids at 15%, wt/wt. Even higher lipid accumulation was exhibited by Nephroselmis sp. KGE1 grown on Bold’s Basal Medium (i.e., which contained KNO₃ = 0.25 g/L as the nitrogen source), with L/x% = 33.0 ± 0.06% (wt/wt) [51]. From the above, it is obvious that lipid production in Nephroselmis is significantly affected by the availability of nitrogen in the growth environment. Therefore, optimization of the culture medium is required. At the same time, the above suggests the different response of strains of the genus to similar types of environmental stresses. In contrast, phosphorus limitation, although not an inhibitory factor for the growth of N. pyriformis PAT2.7, led to the lowest yield among the cultures performed, regardless of the type of bioreactor [8,13,20].

Interestingly, N. pyriformis PAT2.7 seemed to be benefit from nutrient limitation in other aspects of its metabolism, as an increase in the polysaccharide content was observed under nitrogen- or phosphorus-limitated conditions, reaching S/x% = 31.6 ± 2.4% (wt/wt) and S/x% = 21.8 ± 0.7% (wt/wt), respectively. The results of the present study are in agreement with the study of Mastropetros et al. (2023) [21], who recorded polysaccharide contents of 45% (wt/wt) for Nephroselmis sp. grown under nitrogen limitation and high illumination. Moreover, the authors suggested that the application of high salinity and light intensity, as environmental stresses, led to increased polysaccharide production in Nephroselmis.

Regarding protein production, a relatively high content was observed in the N. pyriformis PAT2.7 culture under control conditions, which was lower in comparison with the cultures carried out in other types of bioreactors [8,13,20]. On the other hand, nutrient limitation did not enhance protein synthesis in N. pyriformis PAT2.7. The lower protein content of N. pyriformis PAT2.7 grown under nitrogen or phosphorus limitation confirmed once again that under conditions of environmental stress, microalgae channel energy towards energy conservation and/or growth. However, it is essential to acknowledge that protein production in Nephroselmis can be increased at higher incubation temperatures [21,67].

Lastly, pigment production was generally of the same level in N. pyriformis PAT2.7 cultures and at similar or below the levels reported in other studies [8,13,21,23,69,70]. The carotenoids of Nephroselmis are of particular interest, since they mainly consist of beta-carotene, siphonaxanthin, neoxanthin, and lutein, molecules with proven antioxidant and anti-inflammatory activity [71,72].

4.3. Fatty Acid Composition of Total Lipids and Their Lipid Fractions

The main lipid fraction for the three Picochlorum strains was the lipid fraction G + S, usually followed by the N lipid fraction and then by P. In general, the dominance of the G + S fraction over the total lipids seems to be a shared feature for a plethora of microalgae of different genera, as has been confirmed by various reports [8,13,14,73,74]. This can be attributed to the fact that G + S constitute the primary components (in percentages up to 80–90%) of chloroplasts and thylakoid membrane lipids [63,75,76,77] and phospholipids are the predominant structural lipids in microalgae. The glycolipids (G) of microalgae are considered an important source of omega-3 fatty acids, with a variety of applications, since they are known for their antimicrobial, antiviral, and anti-cancer properties [78,79]. Sphingolipids (S) represent a diverse group of membrane lipids which are characterized by highly conserved evolutionary traits and play a crucial role in cell communication, both with the environment and other cells, as well as in several key cellular processes, such as the control of cell division [80,81].

The level of accumulation of the G + S fraction in the total lipids of a microalgae is influenced by many parameters, such as the composition of the nutrient medium, the photoperiod, the intensity of the light energy provided, the growth phase of the culture, etc. [8,56,82]. For instance, Picochlorum sp. D3 accumulated a significantly lower percentage of glycolipids (≈ 34%) compared with the strains used herein, but overall, the polar lipids of the strain exceeded 75% of the total lipids [14], similarly to the Picochlora used in the present study. It is noted that glycolipids can also play the role of storage molecules and be utilized as a source of carbon and energy, both in heterotrophic (under conditions of paucity of external carbon source) and autotrophic microorganisms [83,84]. Notably, lipid fraction analyses for P. costavermella VAS2.5 revealed that the lipid fraction G + S in cultures grown in mASW.N⁻ and mASW.P⁻ increased from 54.9% of the control conditions to 70.5% and 68.7%, respectively. This observation has also been reported elsewhere. For example, Damiani et al. (2010) [85], growing Haematococcus pluvialis under nitrogen-deficient conditions, recorded an increase in glycolipid accumulation. Similar findings were obtained from the study by Wang et al. (2016) [86] on glycolipid accumulation for the microalgae Chlorella pyrenoidosa grown in growth medium containing nitrogen or phosphorus at various concentrations. The highest rates of glycolipid accumulation in both microorganisms occurred under nitrogen-deficient conditions. Another strain of Chlorella increased its glycolipid content when grown in phosphorus-limiting nutrient medium [87]. In general, a possible explanation for the increase in glycolipid content under nitrogen- and phosphorus-limited conditions is that glycolipids make up for the reduction in other lipid types in order to maintain cell homeostasis. On the other hand, nitrogen deficiency can lead to the reduction in cellular content in thylakoid membranes, the activation of hydrolytic enzymes, and the hydrolysis of phospholipids [88], while phosphorus deficiency can cause a drastic reduction in membrane phospholipids and their replacement by non-phosphorus glycolipids and sulfolipids [89].

Regarding the fatty acid composition of total lipids and the lipid fractions of the examined strains of the genus Picochlorum, the significant synthesis of 18:3(n-3) and 20:5(n-3) was recorded for the P. oklahomense strains and P. costavermella VAS2.5, respectively. The most favorable conditions for 18:3(n-3) synthesis proved to be the control conditions and the least favorable involved the application of a 16:8 photoperiod. The consumption of 18:3(n-3) by humans in reasonable limits can reduce triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein, hence, it is associated with a lower risk of cardiovascular disease and a reduced risk of fatal coronary heart disease [90,91,92]. Intriguingly, the 18:3(n-3) content of the isolated strains was higher than other strains recorded in the literature, e.g., Picochlorum HM1 [7], P. oklahomense UTEX B 2795 (presented as P. oklahomensis UTEX B 2795) [11,15], Picochlorum sp. D3 [14], Picochlorum Azisu1, Picochlorum Azisu2 [55], and P. maculatum [93]. In contrast, the strain used by Dahmen et al. (2014) [53] synthesized 18:3(n-3) at 26.5 ± 1.1% of total lipids. On the other hand, it is of great interest that P. costavermella VAS2.5 synthesized 20:5(n-3) at significant concentrations, especially in the P fraction, where PUFAs are biosynthesized. This PUFA is of particular value, since it is a biosynthetic precursor of prostaglandins-3, which inhibit platelet aggregation, thromboxane-3, and eicosanoids, including leukotriene-5, highlighting its anti-inflammatory and cardioprotective profile [94]. To our knowledge, the presence of this fatty acid has been traced in only one other strain of the genus, though at much lower levels [95]. The high content of P. costavermella VAS2.5 in 20:5(n-3) may explain the very low percentages or even the occasional absence of 18:3(n-3) and 18:4(n-3), according to the mechanisms of PUFA synthesis described in other studies [94,96] and thoroughly discussed in previous studies related to the strains used herein [8,13]. Additionally, P. costavermella VAS2.5 synthesized 16:1(n-7) in significant quantities, a fatty acid to which, among other things, anti-inflammatory properties and the ability to reprogram the intestinal microflora are attributed [97,98]. Notably, only P. costavermella VAS2.5 demonstrated the elevated synthesis of 20:5(n-3) and 16:1(n-7) compared with the control experiment, especially under nitrogen limitation.

In the cultures of M. gaditana VON5.3, when it was possible to determine the lipid fractions, the G + S fraction was found to be dominant. The predominance of G + S lipids over N in members of the genus has been documented elsewhere [8,13,30,31]. However, it should not be neglected that a crucial parameter for total lipid composition seems to be the age of the culture. For example, M. gaditana 1049 was shown to synthesize mainly N lipids; however, this changed after the 16th day of cultivation [99]. The authors interpret this phenomenon as a consequence of a shift in the lipid biosynthesis pathways of aged cultures, redirecting from chloroplasts and other cell membranes mainly towards the production of N lipids. With regard to the lipid composition of M. gaditana VON5.3, the very high percentage of G + S over total lipids is noteworthy, exceeding 70% (wt/wt) when M. gaditana VON5.3 was grown in mASW.N⁻, which can be explained by what was discussed above for Picochlora. Overall, given that G + S are richer in PUFAs, M. gaditana VON5.3 under specific growth conditions appears to be a rather attractive candidate for PUFA production.

M. gaditana VON5.3 produced 16:1(n-7) and 20:5(n-3) in significant quantities, especially in polar lipids, at similar levels compared with the control conditions, in both nitrogen- and phosphorus-limited cultures. As mentioned in the previous sub-section on balanced growth cultures, a similar pattern in total lipids and their lipid fractions was recorded for other Microchloropsis strains of other previously reported studies [30,31,100,101]. Furthermore, M. gaditana VON5.3 presented a significantly higher 20:5(n-3) content compared with the nine Nannochloropsis and Microchloropsis strains used in a study carried out by Ma et al. (2014) [102], where the most productive strain in terms of 20:5(n-3) did not exceed 13% (wt/wt) of total lipids. Moreover, in many cases, the content of M. gaditana VON5.3 in 20:5(n-3) was comparable to the commercial strains that were used by Castejón & Marko (2022) [100] and Abdelkarim et al. (2025) [40] in their studies. The above suggests the ability and suitability of M. gaditana VON5.3 as a source of lipid production of high nutritional and medicinal value.

Lastly, regarding N. pyriformis PAT2.7, interestingly, in contrast to what was recorded for its culture under control conditions and the other microalgae that were examined herein, the dominant lipid fraction was N when this strain was cultured in mASW at pH = 7.5 under constant illumination or in mASW at pH = 8.5 and a photoperiod of 16:8. These findings can be justified by the fact that some microalgae increase their N lipid content under environmental stress conditions, as was probably the case for N. pyriformis PAT2.7. Notably, that was also the case when this strain grew at 20 °C [8], a condition in which polar lipids usually dominate. In general, at low temperatures, the presence of saturated lipids in cell membranes reduces their fluidity and thus negatively affects cellular metabolism. Consequently, low temperatures are expected to stimulate an increased synthesis of unsaturated fatty acids, a feature that could increase survival under such unfavorable conditions [103,104,105]. The notably high content in N lipid reserves is another indication of the ability of this microalga to survive at 20 °C. However, Alboresi et al. (2016) [106] recorded a high N content in M. gaditana, as well, a feature that was attributed by the authors to the high light intensity provided to this microalga. On the other hand, it was not possible to further investigate the total lipid composition in their lipid fractions under nitrogen and phosphorus limitation due to the insufficient amount of extracted lipids.

Regarding the composition of lipids and their lipid fractions in fatty acids, where it was possible to determine them, the composition of mainly saturated fatty acids and the absence of PUFAs was observed. These results give prominence to this strain as a more suitable potential candidate for the biodiesel production industry. Nonetheless, it cannot be ignored that the predominant fatty acid in each case was 16:1(n-7) at outstanding levels. This fatty acid occurred in similar proportions in the N and G + S lipid fractions, while its proportion was reduced by half in the P fraction. The significant production of 16:1(n-7) by N. pyriformis PAT2.7 is considered to be of high importance and possibly opens up new prospects for the exploitation of this microalga.

Nonetheless, lipid biosynthesis depends on the growth phase of the microalgae. For example, the harvested biomass of N. pyriformis PAT2.7 during the late exponential or static phase contained increased amounts of N. Other strains of the genus synthesized 16:1(n-7) (3.9–6.1%, wt/wt) in much smaller amounts [19,67] but also amounts of linolenic acids (18:3(n-6) and 18:3(n-3)), which were not detected in the case of N. pyriformis PAT2.7, which in total approached 15% (wt/wt) of total lipids. However, it is worth mentioning that 18:3(n-6) in other works was detected at 37.7% and 56.4% of total lipids [51,69].

5. Conclusions

All strains used in this study generally produced a satisfactory amount of biomass when grown under control conditions, though higher biomass production was recorded for P. oklahomense SAG4.4 and N. pyriformis PAT2.7 under phosphorus-limited conditions. Conversely, nitrogen limitation emerged as the most unfavorable condition for microalgal growth, highlighting the essential role of adequate nitrogen availability in the growth medium. In addition, the application of a 16:8 photoperiod revealed the pronounced influence of both light intensity and exposure duration on microalgal growth. In contrast, the optimal condition for lipid accumulation for all strains was cultivation at pH 7.5, with phosphorus limitation also proving beneficial in certain cases. Also, the protein and pigment content were usually higher under the aforementioned conditions. Nonetheless, the polysaccharide content of the examined microalgae ranged at similar levels, regardless of the culture conditions. With regard to the composition of lipids and lipid fractions in fatty acids, the PUFAs 20:5(n-3), in the cases of P. costavermella VAS2.5 and M. gaditana VON5.3, and 18:3(n-3), for P. oklahomense, were traced in significant quantities, mostly in the dominant polar lipids, regardless of the culture condition. The exception was N. pyriformis PAT2.7, for which a low lipid composition and a general dominance of N lipids was recorded. At the same time, it is worth mentioning that in the polar lipids of P. costavermella VAS2.5 and M. gaditana VON5.3, 16:1(n-7) was also high, indicating their suitability as a source of lipid production of high nutritional and medicinal value.

Overall, the findings of this study have demonstrated the potential of the examined strains to be exploited under specific culture conditions for commercial purposes, primarily due to their interesting lipid profiles and high protein levels, features of obvious interest for the aquaculture sector. However, since research has shown that the growth capacity and productivity of strains vary between genera, species, and even between strains and is highly influenced by the culture conditions, further optimization is needed with respect to the desired metabolic product.