Biomass Productivity, Amino Acid and Vitamin Composition of Novel Euryhaline Microalga Ava limnothalassea And-Uz-76

Background and Objective: Ava limnothalassea And-Uz-76 is a newly identified indigenous euryhaline microalgal strain isolated from aquatic ecosystems in Uzbekistan. Although its taxonomic identity has recently been established, information regarding the algal biomass productivity and nutritional composition relevant to food biotechnology are still limited. Material and Methods: The strain was cultivated in BG-11, C, CSi, Chu-10, MAV, MDM and MW media. Biomass productivity, crude protein and lipid contents were assessed using standard analytical methods. Free amino acids and water-soluble vitamins were analyzed using high-performance liquid chromatography with diode array detector. All biochemical data were expressed on a dry-weight basis. Results and Conclusion: Biomass production varied depending on nutrient composition. The highest biomass yield (16.0 g l⁻¹) was achieved in C and CSi media, whereas BG-11 media produced 14.6 g l⁻¹ of biomass. Crude protein content reached 31.3–35.9% of dry biomass in C and CSi media. Lipid accumulation increased under nutrient-limited conditions, with the highest value observed in Chu-10 media. Analysis using high-performance liquid chromatography with diode array detector detected nutritionally relevant free amino acids, including glycine, cysteine, proline and asparagine, as well as water-soluble vitamins B2, B3, B6, B9 and C. The indigenous strain Ava limnothalassea And-Uz-76 demonstrated stable biomass production under various cultivation conditions and accumulated proteins with nutritionally important amino acids and vitamins. These findings suggest that Ava limnothalassea And-Uz-76 may represent a promising candidate for further investigation as a sustainable source of protein-rich microalgal biomass and bioactive compounds for further food and feed uses. Keywords: Ava limnothalassea; microalgae; food biotechnology; biomass productivity; crude protein; amino acids; vitamins; euryhaline strain. Introduction Microalgae are valuable sources of proteins, lipids, pigments, vitamins and other bioactive compounds for food industries [1]. The euryhaline microalga of Ava limno-thalassea And-UZ-76, isolated from freshwater and brackish water ecosystems in Uzbekistan, is of particular interest. The A. limnothalassea has been described as a recently identified member of the Chlorellales order and its classification has been verified through molecular and morphological analyses [2]. Microscopic examinations revealed that the vegetative cells of this microalga demonstrated green pigmentation and occur singularly, displaying spherical or elliptical shapes with sizes varying within the microscopic range. The internal cellular structure is characterized by the presence of a single nucleus and a cup-shaped chloroplast, ultrastructural characteristics that are fully consistent with the defining characteristics of the Chlorellales order [3]. In phycology, a coenobium is defined as a non-repeating unit consisting of a fixed number of cells arranged in a constant and interconnected pattern. Unlike general colonies, the cell count and configuration in coenobia are genetically stable. This morphological plasticity highlights the species adaptability to environmental fluctuations. Reproduction occurs primarily through asexual means via autospores (non-motile spores). Within the vegetative cell, protoplast division results in the formation of several daughter cells, which are passive due to the absence of flagella. Following the lysis of the parent cell wall, the autospores are released into the external environment, where they differentiate into independent vegetative cells under favorable trophic and physicochemical conditions (light, temperature and salinity). Within the framework of this study, the effects of hydrophysical factors-specifically water depth, temperature regimes and salinity levels on the biomass productivity and physiological stability of A. limnothalassea were systematically assessed. Previous studies have shown that the growth dynamics and bio-chemical composition of A. limnothalassea are strongly affected by the composition of the nutrient media. Exper-imental results demonstrate that optimizing the physicochemical parameters of the cultivation media allows for the targeted direction of the microalgal metabolic flux. In particular, significant activation of intracellular protein synthesis and maximum biomass yield were observed in the standard BG-11 nutrient media .While BG-11 media served as the control, the growth dynamics of the study object were additionally assessed in seven alternative nutrient media ("C", "Csi", "Chu-10", "MAV", "MDM" and "MW") under laboratory conditions. Clear differences in biomass yield, crude protein content and lipid accumulation were observed in the nutrient media. These results indicate that the biochemical profile of A. limnothalassea And-Uz-76 may be affected by nutrient media composition, with media favoring protein-rich biomass formation and others pro-moting lipid accumulation. Therefore, media optimization can be considered as an important factor for directing the biotechnological use of this strain [4,5]. In particular, the growth performance of indigenous A. limnothalassea strains in various nutrient media, their capacity to produce high biomass and protein-rich biomass and their accumulation of nutritionally valuable metabolites have not been comprehensively investigated. Moreover, data on the amino acid and water-soluble vitamin com-position of this species are still limited [6]. Therefore, the present study aimed to assess the food biotechnology potential of a newly isolated euryhaline microalga A. limnothalassea And-Uz-76 collected from aquatic ecosystems of Uzbekistan. The novelty of this study was linked to the first comprehensive investigation of an indigenous Uzbek strain cultivated in various nutrient media, including assessment of biomass productivity, protein and lipid accumulation and characterization of free amino acids and water-soluble vitamins. The findings provide new insights into the nutritional value and bio-technological applicability of A. limnothalassea as a potential source of functional food ingredients and value-added biomass. Materials and Methods 2.1. Research object and strain origin The unicellular green microalgal strain A. limnothalassea And-Uz-76 (Chlorellales) was used in this study. The strain was isolated in 2023 from artificial freshwater reservoirs in Ulugnor District of Andijan Region, Uzbekistan. The isolation and maintenance of the culture were carried out by researchers from the Tashkent Institute of Chemical Technology, the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan and Tashkent State Technical University named after Islam Karimov. The strain is deposited under the collection number UzRSMMT-432 in the Collection of Rare Microorganisms, Tashkent Institute of Chemical Technology, and in the Collection of Industrially Important Microorganisms, Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan. Based on the collection records, A. limnothalassea And-Uz-76 is the first documented isolate of this species collected in Uzbekistan. Taxonomic identification was based on molecular analysis of the ribosomal DNA internal transcribed spacer region, including ITS1, the complete 5.8S rRNA gene and ITS2. A 519-bp sequence was used as a molecular barcode. The sequence analysis assigned the isolate to A. limnothalassea. The sequence was deposited in NCBI GenBank and the European Nucleotide Archive (ENA) under accession number of PZ035442.1. In this study, no 18S rDNA or *rbcL* sequences were used for taxonomic identification. 2.2. Isolation and culture purification conditions Water samples were collected in 2023 from an artificial freshwater reservoir located in Ulugnor District of Andijan Region, Uzbekistan. Because the taxonomic identity of the target organism was unknown at the time of sampling, the initial isolation was carried out based on morphological and physiological characteristics. After transportation to the laboratory, samples were investigated using light microscope and green, chlorophyll-containing unicellular microalgae with spherical to slightly ellipsoidal cell morphology were selected for further purification. To separate the target microalga from the mixed microbial community, serial dilution and streak-plating techniques were used using solid mineral agar media. The inoculated cultures were incubated at 25 °C ±1 under a 16:8 h light:dark photoperiod and a photon flux density of approximately 45 μmol photons m⁻² s⁻¹. Individual colonies and visually homogeneous algal growth were aseptically transferred to fresh sterile liquid mineral media. The purification procedure was repeated sequentially 5–7 times. At each subcultivation stage, culture morphology was monitored using light and inverted microscopies. Cultures showing the presence of additional algal morphotypes, cyanobacterial cells, or other structures were excluded from further experiments. Only cultures demonstrating uniform cell morphology throughout the purification process were used. As a result, a morphologically homogeneous unialgal culture of A. limnothalassea And-Uz-76 was established and used for further cultivations and biochemical analyses. Taxonomic assignment of the purified isolate was verified using molecular characterization of the ITS1-5.8S-ITS2 ribosomal DNA region, as described in Section 2.1. 2.3. Biomass harvesting and assessment of biomass productivity Biomass was harvested on Day 14 of cultivation, corresponding to the end of the experimental time. The microalgal suspension was concentrated using vacuum filtration and sterile membrane filtration system. The biomass was carefully collected from the filter surface and immediately subjected to freeze-drying to collect dry biomass for further biochemical analyses. To minimize the therma

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