## 4 Discussion  
Despite the wide range of applications, microalgal production is not economically viable yet. Several research pathways have been explored to improve the economics of the process [53]. Among these, recent studies on the effects of bicarbonate in microalgae cultivation aimed to improve biomass production or content of functional molecules [23; 26; 28; 29]. The bicarbonate salt, as an extra source of inorganic carbon for microalgal cultures, has been proposed due to the possibility of being produced by hydration of CO\({}_{2}\) present in polluting fumes, reducing CO\({}_{2}\) emissions into the atmosphere, besides economic advantages [54; 55; 27; 56]. According to [PERSON] and colleagues [57], appropriate concentrations of NaHCO\({}_{3}\) caused the decline of protozoa in cultures, perhaps due to the water efflux affected by rose osmotic pressure. In this study, we focused attention on the short-term physiological and metabolic alterations occurring in algal cells after bicarbonate addition in _Chloella sorokiniana_ cells. According to our data, after 24 h, cultures supplemented with bicarbonate showed a clear increase in the optical density compared to the control. However, the OD increase was not accompanied by an increase in the number of cells. Interestingly, an increase in the cells' diameter in bicarbonate treated cells, even 50-60% higher after 48 h, was observed. This increase in cell diameter size probably could be related to starch or lipid accumulation as cellular carbon storage. In fact, in microalgae, Ci availability represents an important metabolic regulator that controls the carbohydrate and lipid synthesis [25]. According to some studies, lipid accumulation and a larger cellular diameter correlate with a decreased growth [35; 58]. In fact, in some microalgae, bicarbonate addition induced a change in metabolism,  
Figure 8: Principal component analysis (PCA): Distribution of treatments grouped according to different bicarbonate concentration. Control (orange color), 1 g L\({}^{-1}\) (blue color), 2 g L\({}^{-1}\) (green color), and 3 g L\({}^{-1}\) (pink color). shifting the cell from a growth state to a carbon-containing-product formation [24]. Cellular replication considerably stopped at the time of bicarbonate addition in _Chlorella vulgaris_[36]. However, the addition of bicarbonate has not always brought positive effects in microalgae cultures, especially in freshwater species [54]. In fact, adding bicarbonate leads to Na\({}^{+}\) ions rise in the growth medium, and although marine species tolerate them, most freshwater ones do not [54; 59]. In _Chlorella vulgaris_, a supply of a high concentration of bicarbonate (160 mM) inhibited cell growth leading to the formation of numerous colonial cells [35]. Furthermore, according to [PERSON] et al. [22], in _Synechococcus_ sp., an excess of NaHCO\({}_{3}\) caused salt stress, ROS (reactive oxygen species) production, and a decrease in photosynthetic efficiency caused by damage to PSII complex. Our data, however, did not indicate any damage to _Chlorella sorokiniana_ cells treated with bicarbonate, suggesting that the concentrations we used were under the threshold of tolerability. In _Chlorella sorokiniana_, the addition of bicarbonate led to an increase in Chl-_a_ for the entire duration of the experiment, with the highest values in cells supplemented with NaHCO\({}_{3}\) 3 g L\({}^{-1}\). The increase in Chl-_a_ contents per cell in the experimental cultures may be due to the greater availability of inorganic carbon in the growth medium that supports synthesizing the pigment. Since chlorophyll contents represent a valid indicator of the physiological status in microalgae [26; 37], the high levels found in bicarbonate treatments are a signal of good cell health. Moreover, the lower Phaeo:Chl-_a_ ratio in the NaHCO\({}_{3}\) 3 g L\({}^{-1}\) treatment between 24 and 72 h indicates a greater presence of active chlorophyll compared to the control.