## 3 Results and Discussion
### Visible Observation and UV-Visible Spectroscopy of AgNPs Biosynthesis  
#### 3.1.1 Impact of Nitrogen Source  
In this study, _S. obliquus_ was cultivated in BBM medium with six different sources of nitrogen. The cultivation nitrogen source affects microalgal growth through different biochemical pathways that are expected to produce different extracellular active substances [45] and subsequently influence the green biosynthesis of AgNPs. The results for the _S. obliquus_ growth parameters are presented in Table 1. The highest growth was recorded when urea was used as the nitrogen source for _S. obliquus_. The growth status of a microalgal culture reflects the extracellular compounds that are secreted into the medium, including reducing agents that are responsible for silver nanoparticle biosynthesis [12]. The supernatants containing the metabolites of _S. obliquus_ cultivated for 18 days were examined to reduce silver nitrate salt and produce nano-size silver particles. After 24 h incubation with the cultural filtrates, the colorless silver nitrate solutions were changed from yellow to reddish brown in color for all N treatments, but to lesser levels in the cases of urea and ammonium carbonate. Such brown colors have been reported to be characteristic of the formation of AgNPs [17]. The mixtures were scanned by spectrophotometer and the results are illustrated in Figure 1. According to amounts and particle sizes, several peaks were recorded in the range of 380-425 nm corresponding to AgNPs [17; 24]. The highest peaks were detected at 385 and 390 nm for the KNO3 treatment followed by the peaks for the NaNO3 and ammonium sulfate treatments at 380 nm (Figure 1a,d,f). Additionally, the peaks obtained from \begin{table}
\begin{tabular}{l l l} \hline
**Nitrogen Source** & **OD (Abs \(\epsilon_{\text{80 nm}}\))** & **Dry Biomass (g/L)** \\ \hline
**NaNO3** & 2.33 \(\pm\) 0.2 b & 0.63 \(\pm\) 0.013 c \\
**CO(NH4)2 (urea)** & 3.20 \(\pm\) 0.3 a & 1.09 \(\pm\) 0.046 a \\
**(NH4)2 CO3** & 2.66 \(\pm\) 0.2 b & 0.76 \(\pm\) 0.046 b \\
**KNO3** & 1.92 \(\pm\) 0.2 c & 0.48 \(\pm\) 0.008 e \\
**NH4 NO3** & 2.72 \(\pm\) 0.3 b & 0.56 \(\pm\) 0.004 d \\
**(NH4)2 SO4** & 0.84 \(\pm\) 0.1 d & 0.26 \(\pm\) 0.001 f \\ \hline \end{tabular}
\end{table} 
Table 1: Changes in growth parameters of _S. obliquus_ with different nitrogen sources at end of incubation period (18 days). Data are expressed as mean values \(\pm\) standard deviation (n = 2). (NH\({}_{4}\))\({}_{2}\)CO\({}_{3}\) and NH\({}_{4}\)NO\({}_{3}\) were recorded at 380 and 425 nm, respectively, and were obviously smaller (Figure 1c,e). It is worth mentioning that the supernatant from the urea treatment did not yield any peaks in the range of AgNPs detection (Figure 1b). These results could be explained according to the superior role of KNO\({}_{3}\) to induce nitrate reductase production in the cell-free culture supernatant of the 18-day-old _S. obliquus_ culture. Nitrogen is an essential and critical macronutrient affecting microalgal growth and cells' biochemical composition [30; 46]. Most microalgae can utilize various nitrogen sources such as nitrate, nitrite, ammonium (inorganic), and urea (organic) [46; 47]. Nutrient uptake and growth of microalgae indicate its enzymatic activities either interiorly or through the surrounding medium [48]. For this reason, in the present study, nitrogen uptakes from different sources were determined in algal cultures through measurement of residual nitrogen in the supernatant (Figure 2). The consumption rates of nitrogen for the three different forms (i.e., urea, nitrate and ammonia) by _S. obliquus_ were recorded at the log, early-stationary, and late-stationary growth phases. Nitrogen in the supernatants was determined in each treatment as the original applied form (nitrate or ammonium).