This research was conducted from June 2023 to July 2024. Water samples were taken from ITS Dormitory Pond (Figure 1; Table 1). Samples were observed in the Bioscience and Plant Technology Laboratory, Department of Biology, Faculty of Science and Data Analytics, ITS, Surabaya. Microalgae fatty acid profile test was carried out in the Faculty of Pharmacy, Airlangga University, Surabaya.

Four distinct microalgae isolates were obtained from the ITS Dormitory Pond (Table 2). The isolation process involved serial dilution and the streak plate method, repeated until pure isolates were obtained, followed by periodic microscopic observations to confirm the purity of each isolate (Fernandez-Vanenzuela et al., 2021).

The isolate HDB3a was identified as having round cells with diameters ranging from 4 μm to 10 μm. These cells formed small colonies of 4, 8, 16, or more cells, embedded in a transparent mucilaginous matrix. Each cell contained one or more cup-shaped chloroplasts with chlorophyll, giving them a green appearance. Pyrenoids were present within the chloroplasts, playing a role in carbon storage and metabolism (Figure 2, A1). The morphological characteristics of the isolate align with those described by (Bellinger & Sigee, 2015), particularly the spherical shape, mucilaginous colony formation, and cup-shaped chloroplasts, confirming its classification within the genus Dictyosphaerium. (Krienitz & Bock, 2010) also reported that Dictyosphaerium species typically form colonies as a defense mechanism against environmental stress. Additionally, (Bock et al., 2011) found that Dictyosphaerium-like species develop colonies through mucilaginous stalks originating from remnants of the mother cell, suggesting possible polyphyly within the Chlorellaceae family. (Mikhailyuk et al., 2020) further demonstrated the genus’s morphological diversity by identifying Dictyosphaerium-like forms in terrestrial habitats. The presence of this isolate in a pond affected by domestic waste suggested its ability to thrive in nutrient-rich conditions. Its colony formation and chloroplast structures further support its adaptability to environmental changes.

Based on observations, the isolate coded 16HtDB2b had oval-shaped cells with diameters ranging from 10 to 17 μm. The transparent and smooth cell walls lacked any distinct ornamentation or structural modifications. A large, cup-shaped chloroplast occupied most of the cell’s interior, containing chlorophyll pigments and pyrenoids, which functioned as storage centers for photosynthetic products, such as starch. These microalgae exhibited motility, suggesting the presence of flagella, although the flagella were not clearly observed (Figure 2, B1). The morphological characteristics of this isolate align with the description of the genus Vitreochlamys provided by Wehr et al. (2015). Vitreochlamys is a unicellular green flagellate with a smooth, swollen cell wall that separates from the protoplast and features a prominent anterior papilla. Vegetative cells are solitary, biflagellate, and broadly spindle-shaped, pyriform, or ellipsoidal. They typically measure 24 - 34 μm in length and 10 - 22 μm in width, with flagella that are equal to or slightly shorter than the cell length. Two contractile vacuoles are positioned near the base of the flagella. The chloroplast has multiple lobes and contains 4 - 8 similarly sized pyrenoids, which are ellipsoidal, ovoid, or bean- shaped. These pyrenoids are surrounded by two large starch grains and are scattered throughout the chloroplast.

Observations showed that the isolate coded 17HtDB2b had ellipsoid or cylindrical cells, measuring approximately 10 - 30 μm in length and 5 - 15 μm in width. The cell walls featured a complex structure with prominent spines or projections at both ends. The isolate formed colonies of 2 cells to 4 cells arranged in linear or flat formations. These microalgae were non-motile, as they lacked flagella (Figure 2, C1). The morphological characteristics of this isolate are consistent with the genus Desmodesmus, as described by (Bellinger & Sigee, 2015). The spines in Desmodesmus serve as a defense mechanism against microzooplankton predation, as their rigid structure makes ingestion difficult (Trainor, 1996). Additionally, the spines enhance buoyancy, helping the cells remain in the photic zone for optimal photosynthesis (Zhu et al., 2018). The surface morphology of Desmodesmus species, observed under SEM, varied significantly. For example, D. armatus exhibited a warty surface, D. quadricauda had a net-like structure, and D. serratus displayed a star-like pattern. Many Desmodesmus species show phenotypic plasticity, alternating between unicellular and colonial forms, as well as spiny and non-spiny variants. Environmental factors influence these transitions; for instance, D. subspicatus and D. armatus develop spiny colonies under low nutrient conditions, while unicellular forms dominate in phosphorus- or nitrogen-rich environments. When iron availability is low, both colonies and unicellular forms become non-spiny (Shubert et al., 2014).

The isolate coded KD3a consisted of small, round, single cells with diameters ranging from 2 μm to 10 μm. Each cell contained a large, parietal chloroplast occupying most of the cell volume, giving it a green color due to the presence of chlorophyll. The chloroplasts also contained pyrenoids, which facilitate photosynthesis. These microalgae were non-motile as they lacked flagella (Figure 2, D1). The morphological characteristics of this isolate align with those of the genus Chlorella, as described by (Bellinger & Sigee, 2015). Chlorella species are unicellular green algae characterized by spherical or ovoid cells, typically measuring 2 - 10 μm in diameter. Their robust cell walls are composed of cellulose and pectin, providing structural integrity (Takeda, 2004). The genus is distinguished by a single, cup-shaped chloroplast with a prominent pyrenoid, essential for carbon fixation and starch storage. Chlorella is commonly found in nutrient-rich aquatic environments, but can be easily overlooked due to its small size. Its adaptability to various environmental conditions, including polluted waters, makes it a significant genus in both ecological and biotechnological studies (Bellinger & Sigee, 2015).

The harvesting of microalgal isolates was performed prior to conducting fatty acid profile analysis. The optimal harvest time was determined based on the growth curve of the microalgae, specifically during the early stationary phase. This phase is crucial as it marks the onset of metabolite accumulation, including lipids. During the early stationary phase, cell density reaches a constant and maximum level, resulting in the highest biomass concentration (Ferreira et al., 2019). The early stationary phase can be observed through the microalgae growth curve Figure 3. According to the growth curve, the harvest times for the microalgae were as follows: Dictyosphaerium was harvested on day 16, Vitreochlamys on day 15, and both Desmodesmus and Chlorella on day 13. The study results indicated that the microalgal species with the highest total fatty acid content was Dictyosphaerium (30.18%), followed by Desmodesmus (25.72%), Vitreochlamys (21.02%), and Chlorella (4.59%). The fatty acid composition of microalgae isolates from the ITS Dormitory Pond Waters showed significant variation. The fatty acid profile test results indicated that most isolates had high fatty acid content, particularly elaidic acid (C18:1), palmitic acid (C16:0), and stearic acid (C18:0) Table 3.

Dictyosphaerium exhibited the highest saturated fatty acid (SFA) content among the four microalgae species, with a total of 17.57% comprising lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. The species also showed the highest level of monounsaturated fatty acids (MUFA), at 12.28%, predominantly as elaidic acid. Conversely, Dictyosphaerium had a lower content of polyunsaturated fatty acids (PUFA), amounting to 0.33%, primarily as linoleic acid (Figure 4, A). In contrast, (Nayana et al., 2018) reported that Dictyosphaerium contained the highest levels of SFA, specifically palmitic acid (22.64%). The MUFA accounted for 44.24%, including oleic acid (38.08%) and palmitoleic acid (6.16%). The PUFA content was 8.93%, with linoleic acid being the predominant PUFA. The discrepancy between the results of this study and those reported by Nayana et al. (2018) may be attributed to differences in cultivation conditions, which influence the composition of fatty acids produced during growth.

Vitreochlamys exhibited an SFA content of 10.83%, consisting of myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. The MUFA content was 10.19%, predominantly as elaidic acid, with no detected PUFA (Figure 4, B). Research on the fatty acid profile of the genus Vitreochlamys is currently limited. Comparable studies within the family Chlamydomonadaceae can be observed, such as in Chlamydomonas sp., which contains SFA, including myristic acid (2.62%) and palmitic acid (78.35%). This species also has MUFA, like palmitoleic acid (4.26%) and oleic acid (2.48%), and PUFA, such as linoleic acid (3.49%) and arachidonic acid (3.18%). Palmitic acid (C16:0) is the most abundant fatty acid in Chlamydomonas sp., accounting for about 85% of the total fatty acid content, with 9% being PUFA (Morando-Grijalva et al., 2020).

Desmodesmus exhibited an SFA content of 14.19%, which includes lauric acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, and arachidic acid. The MUFA content was 7.39%, primarily as elaidic acid. Notably, Desmodesmus had the highest PUFA content among the four species analyzed, with a total of 4.14%, including hexadecanoic acid, linoleic acid, arachidonic acid, eicosatrienoic acid, and eicosapentaenoic acid (Figure 4, C). In other studies, Desmodesmus was found to contain different compounds under autotrophic (Bourdeau et al., 2017), heterotrophic (Deschênes et al., 2015), and mixotrophic growth conditions (Okpozu et al., 2019).

Chlorella exhibited an SFA content of 1.92%, which included myristic acid, palmitic acid, stearic acid, and arachidic acid. The PUFA content was 2.67%, comprising hexadecanoic acid, linoleic acid, and α-linolenic acid. Notably, no MUFA were detected in Chlorella in this research (Figure 4, D). According to (Rushan et al., 2021), Chlorella cultivated in BG-11, JM, and BBM media displayed typical fatty acid compositions found in microalgal biomass, including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). The highest SFA content was observed in Chlorella cultivated with BBM, followed by JM and BG-11 media. The highest SFA composition in BBM-cultivated Chlorella was attributed to C16:0 (40.74%) and C18:0 (33.33%) of total fatty acid methyl esters (FAME). The highest UFA composition was found in BBM due to the presence of C18:1 (11.11%), C18:2 (7.41%), and C18:3, (3.7%) of total FAME. This shows that using different media will produce different fatty acid contents (Rushan et al., 2021).