Pure strains of Tetradesmus obliquus and Monoraphidium sp. were isolated from two different locations in the Damietta branch of Nile River, Mansoura, Egypt (31°2′25″N 31°22′58″E). Tetradesmus obliquus was cultured in Bold’s Basal Medium (BBM), while Monoraphidium sp. was cultivated in Mannose Binding Lectin (MBL) medium. The selection of BBM and MBL media was based on preliminary experiments conducted to determine optimal growth and lipid accumulation conditions for each strain. BBM supported higher lipid yields in T. obliquus, whereas MBL was more effective for Monoraphidium sp. Both media were prepared using distilled water and sterilized by autoclaving at 121°C for 15 minutes. Cultures were maintained in 5-liter flasks under standard conditions: 25 ± 1°C, a 16:08 h light-dark photoperiod, and illumination of approximately 1.2 klux at the flask surface, provided by cool white fluorescent lamps, over a 28-day cultivation period. To promote homogeneity and prevent sedimentation, cultures received continuous filtered aeration and were manually mixed twice daily. Growth was monitored by daily measurements of optical density at 640 nm using a microplate spectrophotometer (Infinite 200 PRO series, TECAN). The pH of the culture medium was continuously monitored using a pH sensor (DFRobot Gravity) throughout the entire cultivation period.

Morphological identification of the selected microalgal species was performed using both light microscopy and scanning electron microscopy (SEM) at magnifications of 1000×. Light microscopy (ZEISS PRIMO STAR BINOCULAR) was conducted to examine the general cellular morphology, including cell shape, size, and arrangement. For more detailed surface and structural analysis, scanning electron microscopy was employed using a JEOL JSM 6510 system. Sample preparation for SEM followed the protocol described by Echlin (2009). Algal cells were initially fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2) and stored at 4°C overnight. Following fixation, samples were rinsed three times with the same buffer to remove excess fixative. A graded ethanol dehydration series was then applied, involving successive treatments with 50%, 70%, 80%, 90%, and 100% ethanol, each for 15–20 minutes. The final dehydration step used absolute ethanol to ensure complete removal of water. A drop of the dehydrated sample was placed on a carbon-coated copper grid and air-dried using a Sputter Coating Evaporator (SPI Module – Sputter Carbon/Gold Coater) to create a conductive coating. The prepared specimens were then examined under the SEM to obtain high-resolution images for taxonomic comparison with established morphological descriptions.

For molecular identification, genomic DNA was extracted from the algal biomass using a modified CTAB protocol (Hossen et al., 2025). Amplification of the 18S rRNA gene was carried out using PCR with universal eukaryotic primers: the forward primer (5′-AACCTGGTTGATCCTGCCAGT-3′) and the reverse primer (5′-CCTTGTTACGACTTCACCTTCC-3′). The PCR mixture (25 µL final volume) consisted of 2.5 µL of 10× Taq polymerase buffer containing 1 mM MgCl₂, 2 µL of 2.5 mM dNTPs, 1 µL of each primer (10 pmol µL⁻¹), 0.2 µL of Taq DNA polymerase (5 U µL⁻¹), and ultra-pure water. Amplification was performed using a PTC-100 thermal cycler under the following conditions: initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. PCR products were resolved on a 1.5% agarose gel in 0.5× TBE buffer at 120 V for 30 minutes, stained with 0.5 µg cm⁻³ ethidium bromide, and visualized using a Syngene gel documentation system. Fragment sizes were assessed using a DNA molecular weight marker ranging from 100 to 5000 bp (Fisher, Canada). Amplified DNA fragments were purified using a PCR clean-up column kit (Maxim Biotech Inc., USA) according to the manufacturer’s instructions, involving membrane binding, ethanol-based washing, and elution. The purified PCR products were stored at –20°C until further analysis.

Sequencing of the purified products was conducted using the forward primer and the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on a 3130xl Genetic Analyzer (Applied Biosystems). The resulting nucleotide sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis against the NCBI GenBank database to confirm the identity of the isolates. Multiple sequence alignments were carried out using CLUSTALW version 1.83 (Chenna et al., 2003), and phylogenetic relationships were inferred using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) implemented in MEGA version 12 (Kumar et al., 2018), allowing for the placement of the studied isolates within a broader evolutionary context.

To determine the optimal conditions for biomass and lipid production, the growth performance of Tetradesmus obliquus and Monoraphidium sp. was evaluated under different nitrogen-to-phosphorus (N:P) mass ratios (1:1, 1:4, 1:0.5, and 4:1[control]) and varying light intensities (1.2 [control], 2.6, 3.0, and 4.2 klux). The microalgae were cultivated in 250 mL Erlenmeyer flasks containing 100 mL of Bold’s Basal Medium (BBM) for T obliquus and Mannose Binding Lectin (MBL) medium for Monoraphidium sp., respectively. Each flask was inoculated with 20 mL of a two-week-old algal culture with an initial dry biomass of approximately 0.05 g L⁻¹. The cultures were maintained under controlled conditions (25 ± 1°C) with a 16:8 h light: dark photoperiod for a total duration of seven weeks. Growth was monitored daily through direct cell counting using a ZEISS PRIMO STAR binocular light microscope and by measuring optical density at 640 nm using a microplate spectrophotometer (Infinite^® 200 PRO series, TECAN Group Ltd., Switzerland).

Biomass yield was quantified at the end of the cultivation period by centrifuging the cultures at 4000 rpm for 10 minutes, followed by drying the algal pellets at 60°C until a constant weight was achieved. Biomass was expressed as dry weight (mg DW L⁻¹), and the specific growth rate (µ, d⁻¹) was calculated using the formula:

Where W₀ is the initial biomass, W_t is the final biomass, and d is the incubation duration in days.

To determine the effect of nutrient availability, different N:P mass ratios (1:4, 1:1, 1:0.5) were tested against a control ratio of 4:1. Cultures were incubated for four weeks under these conditions, and biomass concentration and lipid content were evaluated. Additionally, the impact of light intensity on algal growth was investigated using a range of illumination levels: 1.2 ± 0.2 klux, 2.6 ± 0.2 klux (control), 3.0 ± 0.2 klux, and 4.2 ± 0.2 klux, provided by cool white fluorescent tubes. Following four weeks of cultivation, dry weight biomass and lipid productivity were measured to assess the influence of illumination. All growth experiments were conducted in triplicate to ensure reproducibility and statistical reliability.

Total lipid content was determined using the Soxhlet solvent extraction method. Dried algal biomass (0.5 g) was mixed with a chloroform–methanol (2:1 v/v) solution and sonicated at 40 kHz for 10 minutes to disrupt cell walls. The resulting mixture was centrifuged at 6,000 rpm for 10 minutes, and the organic phase containing lipids was carefully collected. Extracted lipids were weighed to calculate lipid yield as a percentage of dry biomass (Dayananda et al., 2005). For fatty acid analysis, lipid extracts were subjected to gas chromatography–mass spectrometry (GC–MS) to identify and quantify the fatty acid profile. Analysis was carried out using an Agilent GC–MS system equipped with a capillary column (e.g., HP-88), following standard temperature programming. Identification was based on retention times and mass spectra compared to FAME standards.

The extracted lipids were converted to fatty acid methyl esters (FAMEs) using a two-step transesterification process (Hartman and Lago, 1973). First, saponification was performed by heating 50 mg of lipid with 2 mL of 0.5 M KOH in methanol at 60°C for 1 hour. After cooling, esterification was carried out by adding 2 mL of 14% boron trifluoride in methanol and heating the mixture again at 60°C for 15 minutes. After the reaction, FAMEs were extracted using hexane, dried over anhydrous sodium sulfate, and stored at −20°C until analysis.

The composition of fatty acid methyl esters (FAMEs) derived from algal lipids was analyzed using GC–MS. Analysis was performed with a Thermo Scientific Trace GC Ultra system coupled with an ISQ Single Quadrupole Mass Spectrometer. Separation was achieved using a TG-5MS fused silica capillary column (30 m × 0.25 mm × 0.1 µm film thickness), with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The GC injector and MS transfer line were maintained at 280°C, and electron ionization was carried out at 70 eV. The oven temperature program initiated at 150°C, held for 4 minutes, and ramped to 280°C at a rate of 5°C/min, followed by a final hold for 4 minutes. Samples (1 µL) were injected in split mode. The identification of individual FAMEs, ranging from C14 to C22, was based on comparison of retention times and mass spectral data with entries in the NIST and WILLY libraries integrated into instrument software. Quantification was performed using the relative peak area percentage of each compound.

Following fatty acid profiling, the physicochemical properties of the produced biodiesel were predicted using the Biodiesel Analyzer software, a specialized computational tool developed by Talebi et al. (2014) and validated for assessing biodiesel fuel quality based on fatty acid composition. This software estimated key fuel parameters including density, kinematic viscosity, cetane number, iodine value, acid value, flash point, and pour point. The predicted values were further compared against international biodiesel standards such as ASTM D6751 and EN 14214 to evaluate the suitability of the algal-derived biodiesel for practical fuel applications.

All experimental data were analyzed using one-way analysis of variance (ANOVA) to evaluate significant differences among treatments. Duncan’s multiple range test was employed to separate means at a confidence level of p< 0.05. Statistical analyses were conducted using SPSS software (version 20, IBM, USA).

Tetradesmus obliquus exhibited a typical colonial morphology under light microscopy, forming coenobia of four to eight elongated cells with straight margins and rounded apices. The cells were arranged linearly or slightly curved, and the cell wall appeared smooth without visible ornamentation. SEM analysis confirmed the linear arrangement and revealed more defined ultrastructural details, including the smooth surface and compact coenobial structure ( Figure 1 ).

Monoraphidium sp. displayed a unicellular form with a crescent-shaped cell morphology and tapering ends. Light microscopy revealed individual cells dispersed in the medium, while SEM provided further details on cell curvature and surface texture. The cells lacked visible spines or ornamentation and had a smooth exterior, indicative of the genus ( Figure 2 ).

Molecular identification based on 18S rRNA gene sequencing confirmed the taxonomic status of both isolates. The nucleotide sequence products of algal DNA were analyzed using NCBI-BLAST for confirming the sequences. The obtained sequences of the two algal isolates were submitted to the GenBank and were given accession numbers, Tetradesmus obliquus (GenBank accession no. PV361776) and Monoraphidium sp. (GenBank accession no. PV300550). Phylogenetic analysis placed the isolates firmly within their respective clades, corroborating morphological findings ( Figure 3 ).

The growth kinetics of T. obliquus and Monoraphidium sp. were evaluated under standard conditions (25 ± 2°C, 1.2 klux, 16:8 h light/dark cycle) are presented in Table 1 . The comparative analysis of Tetradesmus obliquus and Monoraphidium sp. over a 28-day cultivation period revealed marked differences in biomass accumulation, lipid productivity, and growth kinetics. Monoraphidium sp. demonstrated superior performance in dry biomass yield, reaching a maximum of 0.177 ± 0.0078 g L⁻¹ at day 28, compared to 0.047 ± 0.0025 g L⁻¹ recorded for T. obliquus at the same time point. Lipid content in Monoraphidium sp. also showed a significant increase over time, peaking at 0.368 ± 0.049 g g⁻¹ on day 28, while T. obliquus achieved a maximum of 0.213 ± 0.028 g g⁻¹.

The specific growth rate (µ) was generally higher in Monoraphidium sp., with the highest rate observed on day 21 (0.132 ± 0.019 d⁻¹), slightly declining by day 28 (0.123 ± 0.015 d⁻¹), whereas T. obliquus exhibited its peak growth rate earlier, at day 14 (0.113 ± 0.002 d⁻¹). Correspondingly, the growth doubling per day (Dd⁻¹) followed a similar pattern, being higher in Monoraphidium sp. (0.191 ± 0.027 d⁻¹ on day 21) than in T. obliquus (0.163 ± 0.003 d⁻¹ on day 14). Generation time (G) was shortest in Monoraphidium sp., particularly on day 21 (5.23 ± 0.73 days), while T. obliquus required more time to double its population, especially on day 21 (10.50 ± 1.75 days).

The impact of varying nitrogen to phosphorus (N:P) ratios on dry biomass and lipid content was evaluated for Tetradesmus obliquus and Monoraphidium sp., as illustrated in Figure 4 . In Tetradesmus obliquus, the dry biomass yield was significantly influenced by the N:P ratio ( Figure 4A ). The highest dry biomass (0.065 g L⁻¹) was observed at an N:P ratio of 1:1, followed by 4:1 and 1:0.5, with the lowest biomass (0.038 g L⁻¹) recorded at the 1:4 ratio. Statistically, the 1:1 ratio was significantly higher than all other treatments (p< 0.05), except 4:1, which showed intermediate values. For Monoraphidium sp., dry biomass followed a different trend ( Figure 4B ). The 4:1 N:P ratio resulted in the highest biomass production (0.085 g L⁻¹), which was significantly greater than that of all other treatments (p< 0.05). The lowest biomass (0.054 g L⁻¹) was found at the 1:4 N:P ratio. In terms of lipid content, Tetradesmus obliquus showed a significant increase at the 1:1 N:P ratio, reaching 34.8% ( Figure 4C ). All other N:P ratios (1:4, 1:0.5, and 4:1) resulted in significantly lower lipid percentages (19.2–22.5%) and did not differ significantly among each other (p > 0.05). Similarly, Monoraphidium sp. exhibited the highest lipid content (47.3%) at the 1:1 N:P ratio ( Figure 4D ), which was significantly greater than all other treatments (p< 0.05). The other ratios showed moderate lipid contents ranging between 34.6% and 37.2%, without statistically significant differences between them. These results demonstrate that optimal N:P ratios differ between species for biomass productivity, while a balanced 1:1 N:P ratio generally favors lipid accumulation in both Tetradesmus obliquus and Monoraphidium sp.

The effects of different light intensities (1.2, 2.6, 3, and 4.2 klux) on dry biomass production and lipid content were examined in Tetradesmus obliquus and Monoraphidium sp., as shown in Figure 5 . For Tetradesmus obliquus, dry biomass was significantly affected by light intensity ( Figure 5A ). The highest dry biomass yield (0.105 g L⁻¹) was recorded at 2.6 klux, followed by 3 and 4.2 klux, which showed moderate but significantly lower values. The lowest biomass (0.056 g L⁻¹) was observed at 1.2 klux. Statistical analysis indicated significant differences among all treatments (p< 0.05), with the exception of 3 and 4.2 klux, which were not significantly different from each other. In Monoraphidium sp., light intensity had a marked effect on biomass accumulation ( Figure 5B ). Maximum dry biomass (0.42–0.43 g L⁻¹) was observed at 3 and 4.2 klux, with no significant difference between these two intensities. A moderate biomass yield was recorded at 2.6 klux (0.32 g L⁻¹), while the lowest value (0.14 g L⁻¹) was at 1.2 klux, significantly different from all other treatments (p< 0.05). Lipid content in Tetradesmus obliquus also responded significantly to light intensity ( Figure 5C ). The highest lipid percentage (41.8%) was obtained at 2.6 klux, which was significantly higher than the 1.2 klux treatment (34.1%) and not significantly different from the 3 klux level. The lowest lipid content was observed at 1.2 klux, while 4.2 klux produced a moderate lipid content (38.5%), significantly different from both the highest and lowest values. Similarly, Monoraphidium sp. showed the greatest lipid content (50.6%) at 2.6 klux ( Figure 5D ), significantly higher than the 1.2 klux condition (44.2%) and comparable to the 3 klux treatment. The lowest lipid yield was recorded at 1.2 klux, while 4.2 klux showed intermediate values (46.8%). Overall, 2.6 klux light intensity provided optimal conditions for lipid accumulation in both species, while biomass production was maximized at higher intensities (2.6–4.2 klux), with species-specific responses.

GC-MS analysis of the chloroform–methanol extracts of Tetradesmus obliquus and Monoraphidium sp. revealed a diverse array of bioactive and lipid-derived compounds ( Figure 6 ). In Tetradesmus obliquus, the dominant compounds included hexadecanoic acid, methyl ester (methyl palmitate), 9-octadecenoic acid, methyl ester (methyl oleate), and octadecanoic acid, methyl ester (methyl stearate). Additionally, notable quantities of 1,2-benzenedicarboxylic acid, diisooctyl ester, a phthalate derivative, and hydrocarbons such as 2,4-dimethylheptane were identified ( Table 2 ). Similarly, Monoraphidium sp. exhibited a comparable compound profile, with methyl palmitate, methyl oleate, and methyl stearate again being the dominant constituents ( Table 3 ). Other significant shared compounds included 1,2-benzenedicarboxylic acid, diisooctyl ester and branched alkanes like 2,6,10-trimethylpentadecane.

GC-MS analyses of the biodiesel derived from Tetradesmus obliquus and Monoraphidium sp. revealed the presence of a range of fatty acid methyl esters (FAMEs), which are essential indicators of biodiesel quality ( Figure 7 ).

In Tetradesmus obliquus, hexadecanoic acid, methyl ester (methyl palmitate) was the most abundant component, accounting for 56.61% of the total peak area, followed by methyl oleate (9-octadecenoic acid, methyl ester) at 22.30%. Other notable compounds included methyl myristate and methyl linoleate, as well as lesser quantities of methyl linolenate and 2,3-dihydroxypropyl palmitate. Both saturated (C14:0, C16:0) and unsaturated (C18:1, C18:2, C18:3) FAMEs indicate a biodiesel profile with balanced oxidative stability and cold flow properties ( Table 4 ).

Similarly, Monoraphidium sp. biodiesel was dominated by 1-hexadecanol, a fatty alcohol derived from palmitic acid, with a peak area of 54.75%, and methyl palmitate at 12.09%. Other major constituents included methyl oleate (7.69%), methyl octacosanoate (4.39%), and methyl lignocerate (4.77%) ( Table 5 ). Both species shared several key compounds, particularly methyl palmitate and methyl oleate.

The predicted biodiesel properties of Tetradesmus obliquus and Monoraphidium sp., as estimated using Biodiesel Analyzer software are displayed in Table 6 . Monoraphidium sp. showed a higher proportion of saturated fatty acids (SFA) at 74.65%, compared to 61.19% in T. obliquus, which contributed to its elevated cetane number (CN = 75.96) and reduced degree of unsaturation (DU = 7.69). Conversely, T. obliquus presented a more balanced profile with 22.84% monounsaturated fatty acids (MUFA) and 1.01% polyunsaturated fatty acids (PUFA), leading to a higher DU (24.86) and improved oxidative stability (OS = 119.35 hours), whereas Monoraphidium sp. recorded zero oxidative stability. The iodine value (IV), an indicator of unsaturation, was notably higher in T. obliquus (22.84) than in Monoraphidium sp. (6.92), correlating with its enhanced oxidative resistance. Despite the superior CN and lower IV, Monoraphidium sp. exhibited significantly higher cold flow properties, including a long-chain saturated factor (LCSF = 17.50), cloud point (30.28°C), and cold filter plugging point (CFPP = 38.50°C), suggesting lower suitability for colder climates compared to T. obliquus, which recorded lower values in these parameters (LCSF = 5.85, CP = 25.78°C, CFPP = 1.90°C). Both species exhibited similar kinematic viscosities (1.15–1.16 mm²/s) and densities (0.71–0.74 g/cm³), with T. obliquus also displaying a slightly higher heating value (HHV = 33.41 MJ/kg). Collectively, these findings indicate that Tetradesmus obliquus offers a more favorable balance between fuel stability, cold flow properties, and energy content, making it a more versatile and climate-resilient biodiesel feedstock. While both microalgae strains are viable candidates for biodiesel production, T. obliquus is characterized by a higher content of conventional biodiesel esters, contributing to better oxidative stability and combustion quality, whereas Monoraphidium sp. is distinguished by a higher proportion of longer-chain saturated and alcohol-based lipids, which may enhance specific fuel attributes such as lubricity and viscosity but potentially limit cold flow performance.