The study of the DNA of microalgae and related microbial communities is key in the development of biotechnology, and provides further opportunities for the production of biologically active compounds. Microalgae have been studied for many years due to their unique biological characteristics, such as high biomass productivity, photosynthesis efficiency, ease of cultivation, and the potential in biodiesel production, the last of which surpasses similar indicators in plants [1,2]. In biotechnology, particularly, microalgae are considered to be promising sources of lipids and secondary metabolites. The study of the DNA of microalgae has significant relevance, because they are important producers of biologically active compounds with antibacterial properties, which means that there is potential application in medicine and pharmaceuticals. Furthermore, studies of microbial communities associated with microalgae contribute to the optimization of cultivation processes and increase the efficiency of production of valuable bio-derived products. One of the main areas of physiological research is the identification of species. However, the lack of noticeable structural features, including of phenotypic differences between species and genera, as well as morphological variance in multiphase life cycles, significantly complicate the identification process [3,4]. Traditional morphological methods are insufficient to distinguish among species with similar external characteristics. Currently, DNA barcoding is recognized as an effective tool for fast and accurate identification of various species [5-6]. The unique advantage of the method lies in its application in the classification of all forms of life. Nonetheless, the lack of a universal molecular marker in the identification of both eukaryotes and prokaryotes remains a significant issue. For example, the mitochondrial COI/COX1 gene encoding the cytochrome C oxidase subunit has been successfully used as a molecular barcode to identify most animals [7]. However, there is no agreed upon universal marker for plants yet. Instead, sections of chloroplast DNA (cDNA) are more often used, including the ribulose-1,5-bisphosphate carboxylase (rbcL) large subunit gene and the trnH-psbA intergenic spacer [8]. The carboxylase/oxygenase (RuBisCO) (rbcL) and maturase K (matK) genes usually have a higher barcoding level compared to other genes. Their use makes possible more accurate distinction between interspecific and intraspecific genetic distances within a group of organisms. The rbcl gene is distributed among most eukaryotic phytoplankton, providing a higher resolution than the 18S rDNA gene [9-11]. Recently, some studies have shown that multiple DNA barcoding of various gene combinations provides a better definition than does a single DNA barcode. For example, sequence analysis of rbkl, tufA and internal transcribed spacers (ITSs) has been used to distinguish among green microalgae, such as Chlorella and Scenedesmus [12]. Some researchers of diatoms have utilized barcoding that applies four different types of genes, including ITSs and conservative sites (CSs), which demonstrates their significant differences, thus making them suitable for species identification [13].

Before conducting polymerase chain reaction (PCR) to identify microalgae, performing exacting DNA extraction and purification is especially key. In order to extract DNA from microalgae, a sufficient amount of biomass, of at least 0.1-0.5 g, should be collected from a single source only after cultivation [14]. Molecular analysis of microalgae solves many problems, including species identification, assessment of genetic diversity, and determination of environmental adaptations. The goals include: improving biotechnology applications, monitoring environmental impacts, and improving taxonomy through DNA barcoding techniques, inclusive of the rbcL, matK and ITS regions. The main challenges are the optimization of DNA extraction, selection of universal genetic markers, and application of advanced molecular tools alongside traditional methods. Particularly, molecular markers play a key role in species identification and biodiversity research. They make possible the study of genetic differences between and within species, which is especially important for complex groups of organisms such as microalgae. The use of markers, among which are ITS, rbcL, and matK, helps to improve the taxonomy, identify new species, and understand ecological adaptation. These methods integrate molecular data with ecological and morphological characteristics to achieve more accurate monitoring of biodiversity [14].

Microalgae are very diverse and can be found in both freshwater and marine environments. Freshwater microalgae include Chlorella and Scenedesmus, which are important for research in biofuel production and environmental monitoring, due to their high biomass productivity. Marine microalgae, including Phaeodactylum and Symbiodinium, play an important role in aquatic ecosystems and have potential applications in biotechnologies such as biofuels and pharmaceuticals. These genera represent only a small portion of microalgae, each of which plays a unique biochemical and ecological role. Understanding their chemical composition, such as the structure of cell walls, is crucial in optimizing DNA extraction methods and advancing the study of these microorganisms for various biotechnological purposes [15].

The chemical composition of the cell walls of microalgae significantly affects DNA extraction. Many species of microalgae, especially diatoms, have complex silicon-based or cellulose-rich cell walls, which can make DNA extraction difficult. The rigid and often thick nature of these walls may require special protocols in order to break down barriers and release intact DNA. For example, diatoms that possess a silicon-based shell often require more aggressive processing, inclusive of enzymatic cleavage or mechanical destruction to successfully extract DNA. Algae with simpler cellulose-based walls, such as some green algae, may be easier to process, but still need optimized methods for the purpose of minimizing degradation or contamination. The type of cell wall, along with other factors, including cell size and age, play a crucial role in the effectiveness of DNA extraction methods [16]. DNA extraction from microalgae is a fundamental step in various molecular biology studies, including genetic analysis, species identification and metabolic studies. There are several methods used to extract DNA from microalgae, each with its own advantages and limitations. Physical methods, such as cell wall destruction, are commonly used to destroy the protective structures of microalgae. Mechanical destruction of cells using a bead mill or mortar and pestle is effective in destroying thick, rigid cell walls. Application of high-frequency sound waves is also used to destroy cell walls and membranes, and is especially useful for smaller or softer cells [16].

Several forms of DNA extraction are available, including: chemical lysis, one of the most common methods, which uses buffer solutions, such as cetyltrimethylammonium bromide (CTAB), and is effective for plant and algae species with thick polysaccharide cell walls, as it destroys cell walls and removes polysaccharide contaminants, but can be toxic and difficult to handle [17]; sodium dodecyl sulfate (SDS), which is a detergent that destroys cell membranes, promoting protein denaturation and DNA release [18,19]; enzymatic treatments, which are used to break down cell walls with less mechanical effort, thus providing a softer alternative; natural enzymes, such as lysozyme, which are used mainly for bacterial-type cell walls, destroying peptidoglycan layers, but are not always effective for all types of microalgae; commercial enzymes, such as Proteinase K, which is an enzyme that breaks down proteins, preventing contamination of the DNA sample [20]; and, commercial kits, such as the Qiagen DNeasy Plant Mini Kit and Sigma-Aldrich GenElute Plant Genomic DNA Miniprep Kit, among others, which offer standardized protocols that simplify the extraction process, and usually include reagents for cell destruction and DNA binding, with the main advantages being reproducibility and time savings, though they can be expensive and are not optimized for all types of microalgae [21]. DNA extraction from microalgae is associated with unique difficulties. Polysaccharide contamination results in a high content of polysaccharides in many types of microalgae, which can alter the DNA sample, thus making difficult the extraction of pure DNA [22]. Inhibitors include secondary metabolites, such as phenolic compounds and proteins, which can inhibit PCR and other subsequent analyses, thus leading to additional purification steps [23].

Molecular markers are important tools in the study of microalgae, because they provide insight into taxonomy, biological diversity, and evolutionary relationships. These markers are widely used in various molecular techniques, inclusive of DNA barcoding, genetic diversity studies, and species identification. Due to its universal presence in all eukaryotic organisms, the 18S rRNA gene is one of the most commonly used molecular markers for microalgae, as it has a high degree of conservatism and provides valuable phylogenetic information. Similarly, the regions of the internal transcribed spacer (ITS) that are located between rRNA genes are variable, and are excellent in distinguishing among closely related species. These markers are necessary for the identification of various microalgae taxa, including both marine and freshwater species [24,25]. Chloroplast genes, such as rbcL, a large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and tufA, a coding protein involved in the synthesis of chloroplast protein, are widely utilized in the study of photosynthetic microalgae, which are more specific to such algae and provide insight into their evolutionary relationships and functional adaptations related to photosynthesis. The rbcL gene, in particular, is a reliable marker for the identification of various algae species, because it participates in the critical carbon fixation process [26].

Furthermore, inter-simple sequence repeat (ISSR)-PCR markers are used to detect genetic variability based on the amplification of sites between simple repeats of sequences, and are highly polymorphic, so do not require prior sequence information, which makes them valuable in studying genetic diversity within and between microalgae populations. Additionally, they are used to assess intraspecific variability, such as in cloned cultures, and interspecific differences, thus providing information on the biological diversity among various microalgae [27,28]. Random amplified polymorphic DNA analysis (RAPD-PCR) is another molecular method used to identify the genetic variability of microalgae, and involves the amplification of random DNA fragments using short primers. Although RAPD-PCR is fast and does not require prior knowledge of the genome, it has limitations in reproducibility and resolution in comparison to other methods, such as ISSR-PCR or sequence-based markers. Despite these limitations, RAPD-PCR can be useful for preliminary studies of genetic diversity and population structure [29,30].

Overall, the utilization of molecular markers allows for species identification, assessment of genetic diversity, and ecosystem monitoring. Those like 18S rRNA and rbcL are crucial for the identification of microalgae species, especially ones that are morphologically similar or lack distinctive physical characteristics, allowing for accurate taxonomic classification. For example, the ITS regions are commonly used to differentiate species in complex groups such as diatoms and dinoflagellates [31]. ISSR-PCR and RAPD-PCR are effective tools for the study of the genetic diversity of microalgae, both in natural populations and under controlled laboratory conditions, allowing assessment of intraspecific diversity, genetic variability, and the influence of environmental factors on microalgae populations. For example, ISSR-PCR markers have been used to assess the genetic diversity of Chlorella and other freshwater algae [32]. Similarly, RAPD-PCR has been applied to the assessment of genetic diversity among marine microalgae, helping in conservation and restoration efforts [33]. In the research with Chlorella species, RAPD-PCR with decamer random primers was used to assess the genetic variability in Chlorella spp [34]. RAPD markers were applied to the amplification of microalgal DNA extracted using the CTAB method. The results showed that primers OPB-01, OPB-11 and OPH-08 exhibited the highest intraspecific variability, with 100% polymorphism, whereas OPH-07 showed the lowest polymorphism, only 13%, at amplicon levels. Despite morphological similarities, a significant genetic polymorphism was observed among Chlorella species. The findings indicate that four RAPD bands are useful in studying the genetic diversity of microalgae, as they allow for differentiation of species. In another study, RAPD markers were used to assess genetic differences among species within three genera of unicellular microalgae: Tetraselmis, Chlorella and Nannochloropsis [35]. Although morphological identification was challenging, due to the similarities among the various strains, RAPD-PCR analysis revealed genetic diversity, the lowest of which was observed between Tetraselmis and Chlorella, at 0.36, and the highest was between Chlorella and Nannochloropsis, at 0.69. Additionally, significant genetic diversity, in the range of 0.58-0.83, was detected within cultured populations of Nannochloropsis. Limited genetic diversity in microalgae cultures may threaten their long-term stability and adaptability under laboratory conditions. Using the genetic fingerprinting method called amplified fragment length polymorphism (AFLP-PCR), genetic diversity among strains of Chlorella vulgaris was examined, along with the identification of cryptic species [36,37]. Different isolated strains of the same species were shown to be genetically distinct, emphasizing the need for careful strain identification in experiments. Further studies showed that AFLP-PCR could be utilized in differentiating pigment mutants of ParaChlorella kessleri, as well as in detecting viral infections in Chlorella sp. The research also addressed issues related to contamination, strain mislabeling, and cryopreservation, which highlights how AFLP-PCR can be used to ensure the genetic integrity of cryopreserved microalgae. Further, the study also examined the optimization of microalgae DNA isolation methods and the use of RAPD-PCR and ISSR-PCR in order to study the genetic diversity of Chlorella sp. and Scenedesmus sp. under laboratory conditions. Molecular methods, including the use of molecular markers, are increasingly being used in monitoring the health of aquatic ecosystems. These methods make possible detection of harmful algal blooms, species invasions, and changes in the composition of microbial communities. For example, DNA markers have been utilized in identifying those responsible for algal blooms, such as toxic dinoflagellates [38,39]. In addition, molecular instruments are used to monitor the effects of environmental stressors, such as pollution or temperature changes, on microalgae communities, providing early warnings of environmental shifts. Next-generation sequencing (NGS) and metabarcoding have revolutionized the study of microalgae, providing fast and accurate identification of species in complex communities. These technologies facilitate high-throughput sequencing of DNA sites, such as ITS and 18S rRNA, which allows researchers to assess biodiversity and understand environmental dynamics [40].

Finally, among the methods that have so far been used to study microalgae, metagenomics provides a comprehensive approach to understanding their biodiversity and functional roles in ecosystems, by analyzing genetic material directly from environmental samples, which makes possible differentiation among both dominant and less common species. The expansion of metagenomic research may provide insight into how microalgae respond to environmental changes, pollution and climatic stressors. Therefore, future studies may further integrate these tools in the analysis of interactions between microalgae and other microorganisms in the aquatic environment. The development of automated systems for DNA extraction and analysis can significantly improve efficiency and reproducibility in microalgae research. Robotic platforms and automated workflows using commercial kits are increasingly being used to process large volumes of samples in environmental monitoring and biotechnological applications [41]. Researchers should focus on adapting these systems to complex samples, such as microalgae with rigid cell walls. Nonetheless, the diversity of microalgae poses challenges in selecting suitable molecular markers for identification and classification. Universal markers that work in broad taxonomic groups will simplify molecular study. Chloroplast genes such as rbcL and psbA, along with ribosomal markers such as 18S rRNA, are promising candidates for such purposes [31]. Additionally, studies should be aimed at increasing the resolution of these markers to distinguish closely related species.

Generally, significant improvements in physical, chemical and enzymatic DNA extraction methods, along with the development of reliable molecular markers, such as ribosomal genes (18S rRNA, ITS) and chloroplast genes (rbcL, tufA), have expanded the collective knowledge of the taxonomy, genetics and biodiversity of microalgae. The use of molecular markers and effective DNA extraction techniques has become transformative for both scientific research and practical applications. These tools make possible accurate identification of microalgae species, assessment of genetic diversity, and monitoring of aquatic ecosystems, in addition to furthering biotechnological applications, such as biofuel production and pharmaceutical development [42]. Such molecular approaches have made a significant contribution to the conservation of aquatic ecosystems by providing accurate monitoring of species composition and environmental health, and have contributed to the development of biotechnology by identifying new microalgae with industrial potential. In the future, researchers might wish to focus on the integration of advanced technologies such as NGS and metagenomics in order to further expand the field, identify sustainable practices for environmental management, and develop commercial products [43].