Tamanna Deswal, Shivani Narwal, Kusum Ahlawat and Rajesh Dhankhar
Abstract
Accurate identification of environmental microalgal isolates is a prerequisite for any subsequent ecological, ecotoxicological, or biotechnological work, however morphology-based identification of small coccoid green algae is frequently unreliable owing to phenotypic plasticity and convergent morphology among genera such as Chlorella, Monoraphidium, Ankistrodesmus, and Raphidocelis. In this study, a unicellular green microalga was isolated from dairy industry wastewater, purified under axenic conditions, and identified using a polyphasic morphological and molecular approach. Genomic DNA was extracted from the purified culture, and a ~1200 bp fragment of the 18S ribosomal RNA (rRNA) gene was PCR-amplified using the universal primers NS1 and NS4, bidirectionally sequenced, and assembled into a consensus sequence. BLASTn analysis against the NCBI GenBank nucleotide database showed maximum identity (99.72%) with Raphidocelis subcapitata strain SAG 12.81 (accession KF673369.1), and Maximum-Likelihood phylogenetic reconstruction (Kimura-2-parameter model, MEGA7; 11 taxa, 1040 aligned nucleotide positions) placed the isolate, designated TDRD01, within a well-supported clade of the family Selenastraceae, sister to the reference R. subcapitata sequence, with a pairwise genetic distance of only 0.001 substitutions per site. Combined morphological and molecular evidence confirmed the isolate as Raphidocelis subcapitata with accession number PZ626732. Dairy wastewater served solely as the environmental source from which the strain was recovered; the present study reports its isolation, purification, and taxonomic characterization.