Chemicals
Analytical-grade chemicals such as Bold’s Basal Medium, ampicillin, Lugol’s solution, Nile red, CTAB buffer, and PCR reagents were sourced from Sigma-Aldrich. The equipment used included a Magnus MLXi Plus microscope, REMI C852 centrifuge, and Lasany Instruments UV-VIS spectrophotometer (Model No. LI 295).

Sample collection, isolation and growth conditions
A microalgal sample was isolated from the stream of Yamuna River at Okhla, Delhi (latitude 28.52° N, longitude 77.28° E). Water samples were collected using the grab sampling method in a sterile white bottle. To reduce the microbial load and facilitate isolation, the sample was diluted with sterile distilled water and transported to the laboratory, where it was processed on the same day. The streak plate method was used to culture the algae in Bold Basal Medium (BBM) in Petri plates. Successive rounds of streaking were performed on BBM plates supplemented with ampicillin to eliminate bacterial contaminants and obtain a purified (axenic) culture. Ampicillin, a β-lactam antibiotic, selectively inhibits bacterial cell wall synthesis without affecting algal cells, thereby aiding in the purification of the algal culture. The resulting axenic culture was maintained in BBM and incubated at 25 °C under continuous illumination (100 lux) with constant shaking at 150 rpm on an orbital shaker.¹⁵

Characterization of P. kessleri
Light microscopy
The algal sample was transferred onto a clean glass slide using a pipette, followed by addition of Lugol’s solution to enhance visibility. A coverslip was gently placed over each sample to prepare a wet mount. The algal cells were examined under a bright-field microscope at 100x magnification, and the features and characteristics of the microalgal isolate PDP_01 were documented. The isolate was morphologically identified as P. kessleri based on its observed characteristics and comparison with the taxonomic descriptions available in AlgaeBase.^22,23 The morphological identification was further confirmed by Dr. U. Elaya Perumal, Scientist, Annakkili Amma Research Institute (AARI), Chennai, Tamil Nadu, India.

Fluorescent microscopy
Intracellular lipid droplets were identified using Nile Red (9-(diethylamino)-5H-benzo[α]phenoxazine-5-one) fluorescence staining. Microalgae biomass was first collected by centrifugation at 250 g for 10 minutes and resuspended in 200 µL of distilled water by vortex mixing. The cell suspension was then treated with a 1% (w/v) Nile Red solution and incubated for 10 min under dark conditions. Excess dye was removed by washing, and lipid accumulation was visualized using fluorescence microscopy within an excitation range of 535-650 nm.²⁴

Molecular identification
DNA extraction
Genomic DNA was isolated from algal samples using a modified CTAB-based method adapted from Doyle.²⁵ The samples were suspended in CTAB extraction buffer and incubated at 60 °C for 1 hrs with intermittent mixing. After cooling to ambient temperature, chloroform:isoamyl alcohol (24:1, v/v) was added, and the mixture was gently mixed for 15 min, followed by centrifugation at room temperature at approximately 16,000 g for 10 min. The aqueous phase was carefully transferred to a new tube and combined with an equal volume of chilled isopropanol to precipitate the DNA, then incubated at -20 °C for 20-30 min. The DNA was pelleted by centrifugation at 4 °C for 10 min, washed with 70% ethanol, and resuspended in TE buffer. DNA integrity and quality were assessed using 0.8% agarose gel electrophoresis prior to PCR amplification.¹⁹

PCR amplification
Amplification of partial 18S rRNA gene sequences was carried out using a thermal cycler. PCR was performed with the forward primer A (5′-AACCTGGTTGATCCTGCCAG-3′) and the reverse primer SSU-inR1 (5′-CACCAGACTTGCCCTCCA-3′), which target conserved regions of the 18S rDNA. Each 20 µL PCR reaction contained 1 µL of genomic DNA template along with the forward and reverse primers. The amplification program consisted of an initial denaturation step at 94 °C for 30 sec, followed by annealing at 55 °C for 30 sec and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min.^22,26

Sequence alignment and phylogenetic analysis
The generated partial 18S rRNA gene sequence was initially examined and manually refined using BioEdit software (version 7.2.5) to eliminate ambiguous regions at the 5′ and 3′ termini. The refined sequence was then compared with reference sequences from the National Center for Biotechnology Information (NCBI) database using the BLAST (Basic Local Alignment Search Tool) algorithm.^22,27 For phylogenetic analysis, the sequence of isolate PDP_01 (GenBank accession OR364517) was aligned with multiple P. kessleri reference sequences and closely related taxa obtained from GenBank. Phylogenetic analysis was performed in MEGA 12 using the Maximum Likelihood method based on the Kimura 2-parameter model with gamma-distributed rates and invariant sites (K2P+G+I), as determined by model selection according to the lowest Bayesian Information Criterion (BIC). Bootstrap analysis was conducted with 1,000 replicates, and sites with less than 95% coverage were excluded by partial deletion. Auxenochlorella protothecoides (MK191970.1) served as the outgroup.

Effect of cultivation factor on the growth behavior of P. kessleri
This study aimed to identify the key factors that influence microalgae growth under controlled conditions. A one-factor-at-a-time experiment was conducted in triplicate, focusing on pH, temperature, photoperiod, and CO₂ sparging duration. The pH levels were varied at 6, 7, 8, 9, and 10, while temperature conditions were set at 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C. The photoperiod was adjusted to different light/dark cycles, including 8:16, 10:14, 12:12, 14:10, and 16:8 hrs. The effect of CO₂ supplementation was evaluated by sparging CO₂ directly from a CO₂ cylinder into the cultures for 1, 2, 3, 4, and 5 min. CO₂ was supplied intermittently twice daily, in the morning and evening, throughout the cultivation period. The CO₂ flow was manually regulated to maintain a low, steady bubbling rate sufficient to ensure uniform gas dispersion without excessive foaming. A flow meter was not used; therefore, the gas flow rate was not quantitatively recorded. All experiments were maintained at a consistent light intensity of 100 lux (Orbit) throughout the study. The cultivation process was carried out in 250 mL Erlenmeyer flasks, each containing 100 mL of Bold’s Basal Medium as the working volume. The experiment lasted 16 days, during which growth was monitored periodically to assess the impact of different parameters on microalgal proliferation.

Growth kinetics
The growth of P. kessleri was tracked by recording the optical density (OD) at 670 nm every three days using a UV spectrophotometer. For biomass quantification, 100 mL culture samples were collected on the sixteenth day and centrifuged at 5000 rpm for 10 minutes to separate the algal biomass. After weighing, the wet biomass was dried at 40 °C in a hot air oven until it reached a stable weight, allowing calculation of dry biomass yield (g/L^-1). The data obtained were used to analyze specific growth rates (µ) and biomass productivity (P) as per equations 1 and 2 respectively. One-factor-at-a-time (OFAT) approach was utilized to determine the optimum conditions for maximum biomass amount.

Specific growth rate (µ) = [In x₂ – In x_{1 /} t₂ – t₁]          ….1

Where x₁ and x₂ denote biomass concentrations (g/L^–¹) measured at time t₁ and t₂.

Biomass productivity: P = [ x_f – x_{i /} t ]        …2

Where “x_f” and “x_i” represent final and initial biomass concentrations (g/L^-1), respectively and “t” represents the cultivation period in days

Validation of growth at optimized condition
After determining the optimum levels for all four input variables, a comparative growth experiment was conducted under optimized and non-optimized conditions. All experiments were performed in triplicate using 250 mL Erlenmeyer flasks with a 100 mL working volume. For the preparation of experimental culture, 1 mL of actively growing P. kessleri inoculum (exponential phase) was aseptically transferred into 99 mL of sterile Bold’s Basal Medium, yielding a total working volume of 100 mL. The optimized conditions were set at pH 8, 25 °C temperature, a 16:8 hrs photoperiod (light-dark), and CO₂ sparging for 2 min twice daily. Optical density (OD) was recorded at three-day intervals, and the dry cell weight (DCW) of the biomass was measured on day 16 to evaluate growth differences between the optimized and non-optimized treatments.

Extraction and estimation of lipid
The extraction and estimation of total lipids were performed from 16 day-old dried algal biomass using the Bligh and Dyer method. The algal culture was centrifuged at 5000 rpm for 10 minutes, after which the resulting pellet was freeze-dried. Following lyophilization, 0.1 g of the dried algal powder was precisely weighed and placed into a 5 mL glass vial. The empty glass bottle was weighed in advance, after which 2 mL of methanol and 1 mL of chloroform were added, and the mixture was refrigerated for 12 hours to facilitate lipid extraction. After incubation, the mixture was centrifuged at 5000 rpm for 10 min. After centrifugation, the lower (bottom) organic phase containing chloroform and extracted lipids was carefully collected. The lipid content was quantified by calculating the ratio of extracted lipid weight to the total biomass weight, expressed as a percentage of dry cell weight using equation 3:

Lipid %= W_I / W_B × 100      …3

where W_l represents the weight of the extracted lipid, and W_B is the total weight of the dry biomass.²⁸

Statistical analysis
All experiments were performed in triplicate, and the results are presented as mean values. Differences between groups were assessed using one-way and two-way analysis of variance (ANOVA) in GraphPad PRISM version 8.0, with a P-value of < 0.05 considered statistically significant.