The pursuit of finding a single solvent capable of solubilizing all target compounds during extraction is challenging. Phenolic compounds, characterized by diverse chemical structures, often form attachments to sugars or proteins in vivo, impacting their solubility across various solvents (Farvin and Jacobsen 2013; Monteiro et al. 2020). Consequently, we assessed the extraction yields of antioxidants from Micractinium sp. under both mixotrophic and heterotrophic conditions using six different solvents with varying polarities. The selection of different solvents aimed to encompass a wide range of polarities, thereby enhancing our understanding of solvent-specific extraction efficiencies and the nature of the compounds being extracted. In our study, methanol and water emerged as the most efficient solvents, yielding the highest extraction percentages under both growth conditions. This finding aligns with previous studies demonstrating the efficacy of methanol and water in extracting bioactive compounds from microalgae (Wang et al. 2009; Jerez-Martel et al. 2017). These results underscore the importance of solvent selection in optimizing extraction efficiency and yield. Furthermore, the significant disparity in extraction yields between methanol and water compared to other solvents highlights the critical influence of solvent polarity on the extraction of bioactive compounds.
The antioxidant capacities of various macroalgae and microalgae have been extensively studied, with notable variations observed across different species and strains (Goiris et al. 2012; Farvin and Jacobsen 2013; Machu et al. 2015). Notably, our study is the first to evaluate the antioxidant capacity of a Micractinium species. Methanolic extracts exhibited the highest antioxidant capacity under both growth conditions, as evidenced by both DPPH and FRAP assays. This observation aligns with prior research highlighting the superior antioxidant potential of methanolic extracts from various microalgae species (Monteiro et al. 2020). The strong correlation between the DPPH and FRAP assays suggests their complementary nature in assessing antioxidant capacity, despite methodological differences (Munteanu and Apetrei 2021). The observed moderate correlation coefficients indicate that while both assays provide valuable insights into the antioxidant potential of microalgal extracts, they may capture different aspects of antioxidant activity.
Comparisons of antioxidant capacities across studies are challenging due to variations in laboratory conditions and methodologies. However, two thermo-tolerant strains, Scenedesmus sp. ME02 and Hindakia tetrachotoma ME03, isolated from the same thermal waters as Micractinium sp., were recently evaluated for their antioxidant capacity (Bulut et al. 2019; Bulut et al. 2023). Our results demonstrated that ethyl acetate and water extracts of Micractinium sp. exhibited higher antioxidant capacity than Scenedesmus sp. ME02 but lower than H. tetrachotoma ME03. The observed differences in antioxidant capacity among the thermo-tolerant strains can be attributed to genetic variability, which influences the production and composition of antioxidant compounds. Each strain has unique metabolic pathways that determine the types and amounts of antioxidants synthesized, affected by enzyme activities and metabolic fluxes. Additionally, strain-specific adaptations to their thermal environments may result in the production of unique antioxidants that confer thermal stress protection. The presence and concentration of secondary metabolites, which act as antioxidants and are often species-specific, further contribute to the variations in antioxidant capacity (Gauthier et al. 2020; Coulombier et al. 2021).
Microalgae cultivation methods, including autotrophy, heterotrophy, and mixotrophy, significantly influence their biochemical content. In this study, we evaluated the antioxidant activity of Micractinium sp. cells grown under mixotrophic and heterotrophic conditions. The notable contrast in antioxidant capacities between these samples underscores the impact of cultivation mode on the biochemical composition and subsequent antioxidant properties of Micractinium sp. Heterotrophic cultivation, particularly when utilizing molasses and vinasse as carbon sources, proved advantageous for achieving higher biomass and lower costs. Micractinium sp. demonstrated adaptability to various growth and temperature regimens, further influencing its biochemical content (Onay et al. 2014; Engin et al. 2018c).
Surprisingly, limited information exists on how heterotrophic growth affects the antioxidant capacity of microalgae compared to other cultivation conditions. In a previous study, the antioxidant activities of Chlorella vulgaris and Scenedesmus obliquus across autotrophic, mixotrophic, and heterotrophic conditions were compared (Shetty and Sibi 2015). Consistent with our findings, methanolic extracts of both microalgae exhibited reduced antioxidant potential during heterotrophic growth compared to autotrophic and mixotrophic conditions. This decline in antioxidant activity under heterotrophic conditions aligns with the understanding that environmental factors, including light and ultraviolet exposure, along with internal processes such as photosynthesis, generate ROS. To counteract oxidative damage from ROS, microalgae produce antioxidants as a defense mechanism. Importantly, microalgae cultivated under mixotrophic conditions are exposed to light and rely on both photosynthesis and an additional carbon source in the culture medium for energy. Consequently, it is plausible to expect a higher antioxidant capacity in mixotrophic cultures compared to heterotrophic growth conditions due to the increased antioxidant activity prompted by the combined effects of photosynthesis and light exposure. This discovery emphasizes the complex relationship between microalgae cultivation methods, environmental influences, and their antioxidant responses, providing insight into the subtle changes in their biochemical processes across different growth environments (Engin et al. 2018b; Gauthier et al. 2020; Abreu et al. 2022).
Studies on other microalgae, such as Tetraselmis suecica and Hindakia tetrachotoma ME03, have highlighted their antioxidant activity and potential applications in the cosmetic and biotechnological industries (Sansone et al. 2017; Bulut et al. 2023). The present investigation into Micractinium sp. ME05 extracts expands the limited knowledge of the in vitro antioxidant activity of green microalgae, further supporting their potential for biotechnological applications.
The evaluation of total phenolic contents (TPC) in Micractinium sp., cultivated under varied growth conditions and extracted using different solvents, provided insights into the phenolic composition of the microalgal biomass. Consistent with prior research, Micractinium sp. extracted with polar solvents exhibited higher concentrations of phenolic compounds compared to nonpolar hexane extracts, regardless of the cultivation method (Goiris et al. 2012; Bulut et al. 2019). Methanolic extracts showed the highest TPC, aligning with previous studies suggesting the effectiveness of methanol in extracting phenolic compounds from microalgae (Safafar et al. 2015). Interestingly, hexane extracts displayed the lowest TPC across both growth conditions, indicating the limited capacity of this solvent to extract phenolic compounds from Micractinium sp. Additionally, our study identified a higher phenolic content in Micractinium sp. compared to Scenedesmus sp. ME02 in ethyl acetate and water extracts, revealing differences in the composition of these two freshwater strains isolated from the same thermal flora (Bulut et al. 2019). These findings underscore the critical role of solvent selection in optimizing phenolic extraction efficiency.
The health-promoting properties of flavonoids, commonly found in fruits and vegetables, are extensively documented in scientific literature, yet research on flavonoid content in microalgae remains relatively scarce (Kozłowska and Szostak-Węgierek 2019). However, insights from previous studies have illuminated the presence of a diverse array of flavonoids across different classes of microalgae, albeit in smaller quantities compared to terrestrial plants (Goiris et al. 2014). Methanolic extracts emerged as the most efficient solvent for extracting flavonoids from Micractinium sp., consistent with its effectiveness in extracting other bioactive compounds. Importantly, our findings reveal that Micractinium sp. exhibits a higher total flavonoid content compared to several other microalgae species, as demonstrated by Bulut et al. (2019) and Safafar et al. (2015). This observation underscores the potential of Micractinium sp. as a promising natural source of flavonoids, suggesting its suitability as a potential substitute for synthetic antioxidants in the industry. The relatively higher flavonoid content in Micractinium sp. extracts, particularly when cultivated under mixotrophic conditions, highlights the importance of cultivation strategies in modulating the biochemical composition and potential applications of microalgae-derived products.
The total carotenoid content (TCC) in Micractinium sp. exhibits significant variation across different cultivation modes, with higher concentrations observed in mixotrophic cultures compared to heterotrophic cultures. This study represents the first attempt to compare the TCC of microalgae under different cultivation modes, exploring the dynamics of carotenoid accumulation in response to varied growth conditions. In a previous study, two Micractinium sp. strains, designated as CCNM 1006 and CCNM 1041, were evaluated for their total carotenoid contents. Both strains displayed slightly higher quantities of total carotenoids compared to Micractinium sp. As part of a broader study encompassing 57 distinct microalgae strains, Micractinium sp. fell within the medium range concerning its TCC (Paliwal et al. 2016). The pivotal role of carotenoids in microalgae involves safeguarding chlorophylls from the detrimental effects of light exposure by scavenging ROS (Sathasivam and Ki 2018). Our findings indicate a higher accumulation of carotenoids in mixotrophic microalgae compared to cultures grown heterotrophically. This underscores the vital role of carotenoids in responding to light exposure, a phenomenon crucial for mitigating oxidative stress through ROS scavenging. Importantly, our study marks the first attempt to compare the TCC of microalgae grown under distinct cultivation modes, offering valuable insights into the dynamics of carotenoid accumulation in response to varied growth conditions.
Despite the well-established role of phenolic compounds in plant antioxidant capacity, their contribution to microalgal antioxidant potential remains debated (Li et al. 2007; Shetty and Sibi 2015; Bulut et al. 2019; Bulut et al. 2023). The diverse nature of microalgal antioxidants collectively contributes to their overall antioxidant capacity. Therefore, we investigated the profile of phenolics, and β-carotene present in the extracts. This study stands out as the first to quantify individual phenolic compounds in a Micractinium species and compare their relative quantities under distinct cultivation modes. Twelve phenolic compounds, categorized into flavonols, benzoic acid derivatives, and cinnamic acid derivatives, were quantified in methanol, acetone, and ethyl acetate extracts from both mixotrophically and heterotrophically grown microalgae.
In this comparative exploration, Chlorella pyrenoidosa, another freshwater green microalga, exhibited considerably lower concentrations of gallic acid compared to Micractinium sp., showcasing the distinct composition of these two species (Machu et al. 2015). 4-hydroxy benzoic acid content (20 µg.g− 1 sample) was higher in C. pyrenoidosa compared to mixotrophic Micractinium sp. (approximately 2 µg.g− 1 DW in methanol extract) but much lower than in heterotrophically grown culture (approximately 400 µg.g− 1 DW in methanolic extract). The significant increase in 4-hydroxy benzoic acid content under heterotrophic growth in Micractinium sp. raises questions about the underlying mechanisms governing these variations, especially considering its known antimicrobial properties used in various industries (food, pharmaceutical, and cosmetics).
Interestingly, despite Micractinium sp. displaying a higher total flavonoid content than Scenedesmus sp. ME02, specific flavonoids like quercetin and rutin were found to be significantly lower in Micractinium sp. This suggests the possible presence of other, unexplored flavonoids in Micractinium sp., hinting at the complexity and diversity of its biochemical profile (Bulut et al. 2019). Additionally, the comparison of cinnamic acid derivatives, chlorogenic, and caffeic acid concentrations between Scenedesmus sp. and Micractinium sp. adds another layer to the variations in flavonoid composition within different microalgal species collected from the same geothermal flora (Goiris et al. 2014).
In emphasizing the importance of specific compounds within microalgae, it is crucial to consider the diverse range of metabolites and their potential applications. Major carotenoid groups, including carotenes (such as β-carotene and lycopene) and xanthophylls (like lutein, astaxanthin, and fucoxanthin), each serve distinct roles. The prevalence of β-carotene in green microalgae like Dunaliella salina and Spirulina maxima underscores their nutritional significance, while Haematococcus pluvialis stands out as a key source of astaxanthin—a commercially valuable product renowned for its various health benefits (Sathasivam and Ki 2018; Maoka 2020; Zheng et al. 2023). In our study, we also quantified β-carotene in methanolic extracts of both mixotrophically and heterotrophically cultivated Micractinium sp., noting slightly higher β-carotene content in mixotrophic cultures compared to heterotrophic ones. Despite this minor difference, β-carotene accounted for approximately 2% of the total carotenoids in methanolic extracts of Micractinium sp., highlighting its substantial presence. These findings underscore the influence of cultivation conditions on carotenoid biosynthesis, with light exposure likely boosting β-carotene production in mixotrophic cultures due to its role in photoprotection and light harvesting.
The methanolic extract of Micractinium sp., which demonstrated the highest antioxidant activity under mixotrophic cultivation, underwent further evaluation for its potential to mitigate intracellular oxidative stress and apoptosis induced by H2O2 in MCF-7 cells. Utilizing the DCFH-DA assay, a well-established method for measuring intracellular ROS levels, our study revealed a significant reduction in ROS in a concentration-dependent manner following pre-incubation with the microalgal extract (Oparka et al. 2016). This substantial decrease in ROS levels is particularly noteworthy as it highlights the potent antioxidant capacity of extracts in protecting cells from oxidative damage induced by H2O2, a stable ROS generator known to cause significant cellular damage at elevated concentrations (Zhuang et al. 2017). The concentration-dependent response observed in this study aligns with the notion that higher concentrations of antioxidants can more effectively neutralize ROS, thereby providing greater protection against oxidative stress.
Apoptosis, or programmed cell death, represents a fundamental cellular process crucial for maintaining tissue homeostasis and eliminating damaged or aberrant cells. Dysregulation of apoptotic pathways is closely associated with various pathological conditions, including cancer (Vitale et al. 2023). In our study, we explored the potential of Micractinium sp. extracts in modulating apoptotic responses induced by H2O2, a potent oxidizing agent known to trigger apoptotic cascades in cancer cells (Zhuang et al. 2017). Our findings reveal a significant reduction in apoptotic rates in MCF-7 breast adenocarcinoma cells pre-treated with Micractinium sp. methanolic extracts, suggesting a cytoprotective effect against H2O2-induced apoptosis. This finding suggests that the methanolic extract of Micractinium sp. not only scavenges ROS effectively but also enhances cell survival under oxidative stress conditions. The significant improvement in cell viability and prevention of necrotic and apoptotic cell death pathways underscore the therapeutic potential of Micractinium sp. extracts in combating oxidative stress-related cellular damage.
These findings align with prior research indicating that microalgal extracts possess robust antioxidant properties and effectively alleviate oxidative stress in diverse cell lines (Sansone et al. 2017; Vahdati et al. 2020; Bulut et al. 2023). In a previous study, Bechelli et al. (2011) investigated the cytotoxic effects of algae, including Dunaliella salina extracts, on normal hematopoietic and leukemia cells by Annexin staining, demonstrating a significant reduction in cell viability induced by D. salina ethanolic extracts. Similarly, Karakaş et al. (2019) demonstrated that the cytotoxic effects of extracts from Chlorella protothecoides and Nannochloropsis oculate on human brain glioblastoma and colon colorectal carcinoma cell lines. To the best of our knowledge, the current study marks the first demonstration of in vitro cytoprotective activity in cell extracts from a Micractinium species. Furthermore, while other studies have explored the cytotoxic effects of various algae extracts on different cell lines, this study uniquely demonstrates the in vitro cytoprotective activity of Micractinium species, opening avenues for further investigations into specific bioactive compounds.
The ability of Micractinium sp. extracts to modulate cell death pathways and enhance cellular viability in the face of oxidative stress holds significant implications for biomedical applications. While our study provides valuable insights into the cytoprotective effects of Micractinium sp. extracts against H2O2-induced oxidative stress in breast adenocarcinoma cells, several avenues for future research warrant exploration. Further elucidation of the underlying molecular mechanisms governing the cytoprotective activity of Micractinium sp. extracts, including their impact on apoptotic signaling pathways and cellular redox balance, is essential for fully harnessing their therapeutic potential.
In addition to whole cell extracts, specific bioactive compounds derived from microalgae have been examined for their antioxidant activity on cell lines. For instance, β-carotene extracted from D. salina strongly reduced cell viability of prostate cancer cells (Jayappriyan et al. 2013). Another carotenoid, violaxanthin isolated from D. tertiolecta showed anti-cancer activity on MCF-7 cells (Pasquet et al. 2011). Polyunsaturated fatty acids extracted from Nannochloropsis salina also exhibited in vitro anti-proliferative effect on MCF-7 cells (Sayegh et al. 2016). While these studies highlight the potential of individual compounds, the use of crude extracts is also important. Crude extracts contain a complex mixture of various bioactive compounds that can work synergistically, potentially enhancing their overall antioxidant and cytotoxic effects. This synergism can lead to a more effective mitigation of oxidative stress and inhibition of cancer cell proliferation compared to isolated compounds. Therefore, exploring the bioactivity of crude extracts provides a holistic understanding of their therapeutic potential and can uncover interactions that may be missed when studying single compounds. Micractinium sp. contains a rich profile of fatty acids, which may collectively contribute to its antioxidant activity.
The versatile characteristics of Micractinium sp., including its adaptability to both mixotrophic and heterotrophic conditions, wide temperature range (16°C-50°C), and diverse biochemical composition, position it as an ideal candidate for mass cultivation with promising applications in the nutraceutical and food industries. Our study represents the first attempt to quantify specific phenolic compounds in a Micractinium species and compare their concentrations under different cultivation methods. Significantly, the antioxidant-rich extracts of Micractinium sp. exhibited a notable inhibitory effect on ROS production and apoptosis induced by H2O2 in MCF-7 cells. This discovery provides valuable insights into the relatively unexplored field of in vitro antioxidant activity of green microalgae for potential biotechnological applications. Future investigations focusing on the identification and characterization of specific bioactive compounds derived from Micractinium sp. can further enhance our understanding of its antioxidant activity, both in vitro and in vivo, thus contributing to the advancement of microalgal biotechnology.