## 4 Discussion  
Wastewater application for microalgae culture is a sustainable and low-cost culture medium that can be used for microalgae biomass production. Municipal wastewaters are a complex mixture of organic and inorganic compounds [17]. They are a source of nutrients (i.e., nitrate, nitrite, phosphorus), but can also contain noxious compounds (i.e., pesticides, household chemicals, hormones) that can affect _C. vulgaris_ growth and compromise the microalgae growth [65]. Moreover, previous studies showed that the inoculation of the microalgae from a standard culture medium directly to wastewater can extend the lag phase, resulting in a lower _C. vulgaris_ growth [66]. The existence of lag phases for all the treatments suggested that _C. vulgaris_ needed a period of adaptation to the SE and SR [63]. The duration of the lag phase for each microalgae culture was, however, different. The higher cell density observed in the SE control was expected, because the growth medium used (BBM2N), is an optimized culture medium that is enriched in nutrients that are pivotal for _C. vulgaris_ metabolic processes and growth [67]. As a counterpart, throughout the experiment period, we observed a microalgae growth reduction in the 25%, 50% and 75% SE concentrations, probably due to the lack of limiting nutrients or due to the self-shading effect caused by the cell density [21; 25]. In SE assay, _C. vulgaris_ achieved a lower specific growth rate (0.32 day\({}^{-1}\)) in comparison with the studies conducted by [PERSON] et al. [68], [PERSON] and [PERSON] [63] and [PERSON] et al. [65], in which _C. vulgaris_ exhibited a value of 0.64, 0.52 and 0.62 day\({}^{-1}\), respectively. Concomitantly, [PERSON] and Ormeci achieved a specific growth rate of 0.25 day\({}^{-1}\) in the 63% SE, while in this study we registered a higher value of 0.42 day\({}^{-1}\) in the SR assay. The enhanced specific growth rates in the 25% SR, 25% SE and 50% SE, compared to control, can be an effect of the organic carbon present in the SE and in the SR [65]. For instance, the presence of these organic nutrients can promote the mixotrophic growth of _C. vulgaris_, enhancing the specific growth rate [69]. The chlorophyll content expresses photosynthetic activity, which varied within the different effluent ratios tested, and is in concordance with the cellular density of the cultures in each treatment. The chlorophyll is an essential pigment that acts as a light converter into energy, through the process of photosynthesis [70]. Furthermore, this pigment has several biotechnological applications for several industries, such as food, feed or pharmaceutical [71; 72; 73]. In this context, several studies have been conducted in order to optimize microalgae chlorophyll production [22; 74; 75; 76]. Growth media with lower  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{ \begin{tabular}{c} **Time** \\ **(Days)** \\ \end{tabular}  } & \multicolumn{3}{c}{**N-NH3 (mg/ L)**} & \multicolumn{3}{c}{**Total N (mg/ L)**} & \multicolumn{3}{c}{**Total P (mg/ L)**} \\  & **Control** & **50\% SR** & **100\% SR** & **Control** & **50\% SR** & **100\% SR** & **Control** & **50\% SR** & **100\% SR** \\ \hline
0 & 0.01 a & 22.62 \(\pm\) & 28.14 \(\pm\) & 15.13 \(\pm\) & 76.49  
nitrogen and phosphorus content have been shown to decrease chlorophyll production, while phosphorus-enriched growth media have revealed to be a chlorophyll production promoter [77; 78]. However, it is necessary to consider that the microalgae growth is not only affected by nutrient concentrations, but also by the ratios of C, N and P in the wastewater [63]. Nitrogen is an important and limiting nutrient for microalgae, being necessary for protein, nucleic acid and chlorophyll molecules synthesis [79]. Consequently, lower contents of nitrogen can lead to a significant reduction of chlorophyll production [80; 81]. Phosphorus is equally a pivotal and limiting nutrient for microalgae growth and development.