## 4 Discussion  
### Environmental Parameters and Culture  
Figure 3 shows that there were no significant differences between the values for light (\(p\) value = 0.949), pH (\(p\) value = 0.241), and temperature (\(p\) value = 0.896) taken in the different systems with a 95% confidence interval (significance level = 0.05). The greatest differences in pH were detected between systems R2 and R3 (\(p\) value = 0.218). The light measurements recorded between systems R1 and R2 differed the most (\(p\) value = 0.750). The temperature was very similar in the R1 and R2 systems (\(p\) value = 0.869), and was slightly different from that of the R3 system (\(p\) value = 0.6). The pH values are represented by the black bars. A maximum value of pH = 10.8 was recorded on day 7 for R1, and a minimum value of 9 was recorded on day 13, which represents a variation in pH of 1.8 throughout the experiment. In the case of R2, the extreme pH values were 10.9 and 8.5, on days 7 and 1 of the culture, respectively, which represents an oscillation of 2.4. Finally, for R3, the maximum pH value was 10 on days 7, 6, and 13, and the minimum was 8.7 on day 15, a variation of 1.3. The microalgae in this study are a species that can tolerate these small changes in pH in the culture; however, these changes can modify the growth of the microalgae, as indicated by [36]. pH is one of the most important factors in microalgae cultures because microalgae membranes are not completely impermeable to hydrogen ions and hydroxy groups. Although \(P\). _triornutum_ is a tolerant species, these changes can be leveraged in the culture. A pH increase occurs as a result of the absence of CO\({}_{2}\) input because the ions are pulled out as bicarbonate by the microalgae. Calcium carbonate precipitation occurs when pH reaches 10, and microalgae may be affected [36]. There are studies in the literature that indicate the onset of flocculation (microalgae aggregation) when pH values are higher than 10.5 [37]. However, this may not be the only explanation for the phenomenon. Flocculation was observed only at the beginning and end of the culture experiment, regardless of pH value, according to [38]. In the absence of nitrogen, this was not observed, although the pH values remained lower than the previously mentioned umbral pH values. Figure 6: Hepatoprotective effect (MTT reduction assay) in HepG2 cell line exposed to methotrexate (MTX) for 24 h.  
Irradiance values can only be used to establish whether the culture conditions remain constant in all systems. To use this variation to infer its influence on the culture, it would be necessary to constantly record this parameter, because slight changes in light intensity can lead to changes in culture growth and productivity rates [39]. In various studies, light has been shown to influence not only the growth and cell division of _P. tricornutum_, but also the pigment content (i.e., fucoxanthin). High irradiance levels are harmful to orbital aerated cultures in fucoxanthin production and culture aeration is essential for growth under high light intensity [40]. However, high light intensity can induce photoinhibition and photom damage, leading to cellular death [41]. In _Haematococcus_ sp., _Dunaliella_ sp. and _Muriellosis_ sp., increased primary carotenoid production was detected under high-irradiance conditions. However, light seems to affect fucoxanthin production in _P. tricornutum_ in a different manner. Some studies have reported increased carotenoid production under medium light conditions [40], while others have suggested that the highest accumulation rates occur at low light intensities [42]. The culture temperature remained constant throughout all experiments, and its influence on P. tricornutum has already been described. The highest division rate was registered at 23 \({}^{\circ}\)C, carbon fixation increased in direct relation to temperature (14-25 \({}^{\circ}\)C), and cells tended to have larger chloroplasts above 21 \({}^{\circ}\)C and lower light intensities [43].