## 3 Results and Discussion
### Nutrients Removal  
In this study, nitrogen (in the forms of nitrate and nitrite) and phosphorus (in the form of phosphate) concentrations were monitored within the cultivation time to evaluate the potential of _C. vulgaris_ to uptake these nutrients from a paper industry effluent with different concentrations of both nitrogen and phosphorus. Figure 3 shows the variation of nitrogen and phosphorus concentration in each assay. Regarding nitrogen removal (Figure 3A), this element was readily assimilated by _C. vulgaris_ in the diluted effluents (assays 2-5). In the raw effluent (corresponding to assay 1), a two-day delay was observed in nitrogen assimilation, which may be related to the adaptation of the microcalga to these conditions. Regarding the assimilation patterns observed in assays 2-5, these were approximately linear for assays 2-4, with nitrogen concentration decreasing gradually during the cultivation time. On the other hand, in assay 5, corresponding to the more diluted effluent experiments, nitrogen concentration decreased until the seventh day of culturing and then it was maintained approximately constant. This behavior may be attributed to a decrease in photosynthetic activity, as nitrogen concentration decreased, and explains the lower biomass concentrations achieved in assay 5 when compared to the one obtained in assay 4 (according to Table 2, \((231\pm 31)\) mg\({}_{\rm dw}\) L\({}^{-1}\) and \((249\pm 14)\) mg\({}_{\rm dw}\) L\({}^{-1}\), respectively). Also, at the end of the cultivation time, nitrogen concentration remaining in cultures corresponding to assays 4 and 5 was approximately the same (\((2.81\pm 0.05)\) mg\({}_{\rm N}\) L\({}^{-1}\) and \((2.6\pm 0.2)\) mg\({}_{\rm N}\) L\({}^{-1}\), respectively), indicating a limitation of this nutrient in the last days of assay 5. As for nitrogen concentration, phosphorus concentration also decreased within the cultivation time (Figure 3B), but in a lesser extent, which is related with microalgal nutritional requirements, as given by its typical elemental biochemical composition: CO\({}_{0.48}\)H\({}_{1.83}\)N\({}_{0.11}\)P\({}_{0.01}\)[24]. The reduction observed in nitrogen and phosphorus concentration in the studied effluent (raw or diluted) shows that _C. vulgaris_ can promote an efficient uptake of both nutrients. However, except for nitrogen concentration in assay 5, total depletion of these nutrients did not occur after the 11 days of culturing, reiterating what was stated in relation to cell growth, that the cultures could be extended for an increased period to further improve nutrients removal efficiencies. Another similarity with the microalgal growth parameters already described is the higher variations in nitrogen and phosphorus concentrations observed in the experiments where the effluent was previously diluted (assays 2-5), which indicate that these conditions were more favorable for _C. vulgaris_ photosynthetic activity. Nitrogen and phosphorus removal parameters are presented in Figures 4 and 5, respectively. As with microalgal growth parameters, a general increase in nutrients removal efficiencies was observed from assay 1 to 5, with values ranging from \((24\pm 10)\%\) to \((80\pm 4)\%\) for nitrogen (Figure 4A) and from \((13.0\pm 0.9)\%\) to \((54\pm 1)\%\) for phosphorus (Figure 5A). However, Figure 4A shows that there was no statistical difference (\(p>0.05\)) in nitrogen removal efficiency between assays 4 and 5, which can be explained by the low concentration achieved in the assay 5 (the one corresponding to the most diluted effluent) that might have been limiting for microalgal growth. In fact, according to Table 2, maximum biomass concentration achieved in assay 4 was higher than that in assay 5, indicating that the highest dilution applied in this study may have contributed to nitrogen limitation to _C. vulgaris_, with effects on their growth and nutrients removal parameters.