## 2 Materials and Methods
### Paper Industry Effluent and Culture Conditions  
Effluent from a Portuguese paper company, collected after the secondary treatment step, was characterized (Table 1) and employed as a culture medium for microalgal growth. The methodology adopted for effluent characterization was the following: (i) COD and turbidity were determined according to the Standard Methods for the Examination of Water and Wastewater [15] (through the 5220-D and 2130-B tests, respectively); (ii) total dissolved carbon (TDC), dissolved organic carbon (DOC) and inorganic carbon (DIC) were determined using an organic carbon analyzer (TOC-V\({}_{\text{CSN}}\), Shimadzu); and (iii) chlorides, sulfates, nitrates, nitrites and phosphates were determined through ion chromatography (ICS-2100, Dionex). Due to the low concentration of nitrogen in the effluent, when compared with typical nutritional requirements of microalgae, the effluent was supplemented with NaNO\({}_{3}\) to achieve N:P molar ratios ranging between 6:1 and 9:1. Ratios between 5:1 and 30:1 have been considered adequate for several microalgal species [16; 17].  
Batch experiments were performed in 1--L borosilicate glass flasks (VWR, Portugal) with a working volume of 950 mL for 11 days. The raw effluent (assay 1) and four different dilutions  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Parameters** & **Values** & **Unit** \\ \hline Turbidity & 1.55 & NTUa \\ pH & 7.02 & - \\ Dissolved organic carbon (DOC) & 296 & mg L\({}^{-1}\) \\ Total dissolved carbon (TDC) & 369 & mg L\({}^{-1}\) \\ Dissolved inorganic carbon (DIC) & 72.5 & mg L\({}^{-1}\) \\ Soluble chemical oxygen demand (COD\({}_{\text{S}}\)) & 323 & mg L\({}^{-1}\) \\ Chlorides (CI\({}^{-}\)) & 671 & mg L\({}^{-1}\) \\ Sulfates (SO\({}_{4}\)\({}^{2-}\)) & 808 & mg L\({}^{-1}\) \\ Phosphate–phosphorus (PO\({}_{4}\)–P) & 12.3 & mg L\({}^{-1}\) \\ Nitrate–nitrogen (NO\({}_{3}\)–N) & 8.73 & mg L\({}^{-1}\) \\ Nitrite–nitrogen (NO\({}_{2}\)–N) & 3.42 & mg L\({}^{-1}\) \\ \hline \hline \end{tabular} \({}^{\text{a}}\) Nephelometric turbidity unit.  
\end{table}
Table 1: Physicochemical characterization of the paper industry effluent used in this study.  
with freshwater (assays 2-5) were used as the culture medium for microalgal growth, with nitrogen concentrations (corresponding to the sum between nitrate- and nitrite-nitrogen) ranging between 12.7 mg\({}_{\text{N}}\) L\({}^{-1}\) and 34.2 mg\({}_{\text{N}}\) L\({}^{-1}\) and phosphorus concentrations (phosphate-phosphorus) ranging between 4.01 mg\({}_{\text{P}}\) L\({}^{-1}\) and 12.3 mg\({}_{\text{P}}\) L\({}^{-1}\). The medium was inoculated with 250 mL of _C. vulgaris_ inoculum to obtain an initial biomass concentration of \(\sim\) 68 mg\({}_{\text{dw}}\) L\({}^{-1}\). The cultures were continuously exposed to: (i) photosynthetically active radiation between 30-40 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\), using a 34-W white led panel; and (ii) atmospheric air filtered with 0.45-\(\mu\)m nylon membranes (Specanalitica, Portugal), injected at \(\sim\) 90 L h\({}^{-1}\), using Trixie AP 180 air pumps (Trixie, Tarp, Germany). The experimental setup is shown in Figure 1. Two independent experiments were performed for each assay.