This study used three Spirulina isolates: the brackish-water strain JPR from Jepara, Central Java, and two freshwater strains, BGR from Bogor, West Java and MRP from the Merapi area, Special Region of Yogyakarta. The research was conducted in the Biology Laboratory, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta, Indonesia.

The morphology of spirulina can be seen in Figure 1. Where images a (BGR) and c (MRP) are spirulina cultivated in fresh water, whereas image b (JPR) is spirulina cultivated in brackish water.

Observations show that there are differences between Spirulina strains living in freshwater and brackish water. The BGR and MRP strains have trichomes that tend to be linear with slight curves, whereas the JPR strain has more convoluted or spiral-shaped trichomes with relatively large spacing between the coils. This morphological difference is influenced by environmental conditions such as light intensity, pH, salinity, and aeration during cultivation (Jung et al., 2021). Freshwater has a salinity of less than 0.5 ppt, while brackish water tends to have higher salinity, less than 30 ppt (Ujwala, 2025). Freshwater has a more neutral pH compared to brackish water; freshwater pH is 7, while brackish water tends to be more alkaline, ranging from 7.7 to 9 (Supriatna et al., 2020). Consistent with the study by Hong et al. (2023), Spirulina exhibits a high adaptability, marked by morphological changes in response to salinity stress. Spirulina living in water with higher salinity experiences shorter trichomes due to fragmentation and looser spiral density. This also aligns with the research by (Nosratimovafagh et al., 2023), which states that increased water salinity thickens the outer spiral sheath, reduces flexibility, and decreases the spiral diameter in Spirulina, thereby increasing the spacing between coils (screw pitch length). Besides the spiral form, Spirulina can undergo morphological conversion into linear filaments, usually influenced by environmental factors and nutrients (Lijassi et al., 2024).

a. DNA Extraction Results

The visualization results of Spirulina DNA extraction can be seen in Figure 2.

Based on the results above, it is known that the three strains have fairly good genomic DNA bands. Strains BGR and JPR have relatively thick DNA bands, whereas strain MRP shows a slight smear on its DNA band. The thickness of the DNA bands indicates the concentration level of DNA in the sample; the thicker the DNA band, the higher the DNA concentration (Alfaruqi, 2021). The presence of smearing on the DNA band is caused by contaminants such as proteins and residual solution during the isolation process (Iqbal et al., 2016).

b. The results of DNA amplification

The results of the spirulina DNA amplification can be seen in Figure 3.

Based on electrophoresis, the PCR technique using the designed primers successfully amplified the 16S rRNA gene from Spirulina. The presence of a DNA band around 500 bp indicates that the genomic DNA of Spirulina was successfully extracted as a template. The study by (Johnson et al., 2022) showed that amplification of the V1–V3 region of the 16S rRNA gene using primers 27F–519R produced fragments approximately 450–550 bp in size, which were effective for identifying and analyzing the phylogenetic relationships of cyanobacteria. The successful amplification of the 16S rRNA gene indicates that the primers used were specific to the target gene and that the PCR amplification proceeded well. Furthermore, no non-specific bands or signs of DNA degradation were found, demonstrating that the primers have high specificity for the conserved region of the 16S rRNA gene.

c. DNA Sequencing Results

The DNA band sequencing results were used to determine the DNA sequence, from which the identity of the spirulina strain could be identified through BLAST analysis. BLAST (Basic Local Alignment Search Tool) is a bioinformatics method used to compare nucleotide or protein sequences with sequences available in databases (Strover & Clavalcanti, 2017). BLAST enables the identification of bacterial species, genus, or phylum based on genetic sequence similarity, especially in gene markers such as the 16S rRNA gene. BLAST analysis of the spirulina strain showed a very high similarity level, with an identity percentage around 99%. The BLASTn analysis results of Spirulina are shown in Table 1.

The results show that the Arthrospira JPR and MRP isolates have the closest homology similarity to the Arthrospira sp. IAQUASC-C0001 species, with percentages of 99.34% and 99.12%, respectively. Meanwhile, the Arthrospira BGR isolate has the closest homology similarity to Arthrospira fusiformis strain AICB 668 16S ribosomal RNA gene, partial sequence, with a similarity percentage of 99.78%. The high identity percentage (>99%) indicates a close phylogenetic relationship with the species found in the database (Aristya et al., 2025). The similarity percentage in this study is lower compared to the research by (Szubert et al., 2021), which stated that spirulina found in the Baltic Sea with codes CCNP1310 and 06S082 showed 100% similarity with S. subsalsa PD2002/gca and S. subsalsa KD 01, indicating similarity above 97%. (Lin et al., 2023) stated that bacterial identification at the species level using the 16S rRNA gene requires a higher sequence similarity threshold, namely above 99% or >99.5%. This is used to improve the accuracy of taxonomic determination.

d. Phylogenetic Analysis

The phylogenetic analysis can be seen in Figure 4.

Based on Figure 4, the 16S rRNA gene sequences show a phylogenetic relationship among three isolates, namely BGR, JPR, and MRP, with several Arthrospira and Limnospira species from GenBank. These three isolates form the same clade and are separated from the Limnospira group. The bootstrap values obtained are relatively high, ranging from 97 to 100, indicating strong confidence in this grouping. Such high bootstrap values suggest that the three isolates have a very close genetic relationship with each other. (Puspitasari et al., 2025) state that the higher the bootstrap value, the greater the accuracy of the tree topology resulting from the reconstruction.

Strains BGR, JPR, and MRP show the highest phylogenetic closeness to Arthrospira sp. IAQUASC-C0001 and Arthrospira fusiformis strain AICB 668, consistent with the BLAST analysis results (Table 1), which show sequence similarity exceeding 99%. Limnospira platensis and Limnospira maxima are placed in a different clade, indicating taxonomic separation between the genera Arthrospira and Limnospira. Although BLASTn analysis (Table 1) shows the highest sequence similarity to Arthrospira species, the phylogenetic reconstruction (Figure 4) places all isolates within the Limnospira clade. This discrepancy is likely due to recent taxonomic revisions, where several species previously classified as Arthrospira are now assigned to the genus Limnospira(Sinetova et al., 2024). Therefore, based on phylogenetic inference, the isolates are more appropriately identified as Limnospira. This grouping pattern shows that the isolates studied are genetically more closely related to the genus Arthrospira than to Limnospira. This supports the identification of the isolates as members of the genus Arthrospira. This aligns with the research of (Zhang et al., 2022), where BLAST results showed that the 16S rRNA sequence similarity of the study isolates with Arthrospira in GenBank was more than 99%, indicating that Arthrospira isolates have high genetic homology and form a single clade in phylogenetic analysis. This study shows that the observed morphological variation more likely reflects phenotypic responses to different environmental conditions rather than significant genetic differences. (Emam et al., 2025) state that spirulina with diverse morphological forms still has a high genetic similarity of around 95%, based on molecular marker analysis