## 3 Results and Discussion
### Visible Observation and UV-Visible Spectroscopy of AgNPs Biosynthesis
#### 3.1.2 Impact of Microalgal Growth Stage  
The aging of microalgae can be a significant factor to the production of their metabolites and to their reduction systems. Therefore, the metabolite composition in the culture filtrate is expected to differ by cultivation time [26]. Microalgae require a long time for desirable cell growth relative to bacteria. In the present study, 6-, 12- and 18-day cultures were regarded as representatives for the log, early- and late-stationary growth stages, respectively [28, 50]. As noted earlier, reductase enzymes were expected to play the main role in the extracellular biosynthesis of AgNPs. The production level of such enzymes as well as other reduction systems varied largely according to the growth stage of the microalga and could be improved via utilization of nitrate-N. Hence, reductase enzyme was considered in the study of the influence of microalgal growth stage on extracellular biosynthesis of AgNPs. Samples were collected at 6-day intervals from each nitrate treatment, and the supernatants were used for the green synthesis of AgNPs. The results obtained for nitrate reductase activity are represented in Figure 3. In the ammonium sulfate treatment, there was no nitrate in the medium, and so no nitrate reductase was detected, but sulfate reductase had been expected to be secreted [51]. On the other hand, the application of nitrate as the N-source induced reductase release in the culture supernatant of _S. obliquus_. However, the reductase activity varied according to both nitrate salt and the algal growth stage. The highest reductase activity was recorded when KNO\({}_{3}\) was applied as the sole N-source, and its values increased with aging of the algal culture. In this case, the nitrate reductase activity reached 49, 70, and 120 U after 6, 12, and 18 days of algal cultivation, respectively (Figure 3). Similar results were observed for the NaNO\({}_{3}\) and NH\({}_{4}\)NO\({}_{3}\) treatments, the values being slightly lower than that for KNO\({}_{3}\). The nitrate reductase enzymes were induced by application of nitrate-N sources, while ammonium-N sources repressed nitrate reductase activities [52, 53, 54]. Figure 2: Residual nitrogen (% from initial) in _S. obliquus_ growth medium throughout cultivation period (18 days) (**a**) NaNO\({}_{3}\); (**b**) urea; (**c**) (NH\({}_{4}\))\({}_{2}\)CO\({}_{3}\); (**d**) KNO\({}_{3}\); (**e**) NH\({}_{4}\)NO\({}_{3}\); (**f**) (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\). Data are expressed as mean values \(\pm\) standard deviation (n = 2). Figure 4 shows the gradual synthesis of the silver nano-form in response to the different nitrate nitrogen sources and cultivation stages. The formation of the silver nano-size in the cultural filtrates of the nitrate as well as sulfate (ammonium sulfate) treatments showed obvious increases with cell aging. This increase was almost linear with the reductase increment. However, the supernatants collected from the cultures fed on KNO\({}_{3}\) produced more nano-silver than in the case of NaNO\({}_{3}\) or (NH\({}_{4}\))NO\({}_{3}\). The absorbance curve indicating the AgNPs' use of the culture supernatant from the KNO\({}_{3}\) treatment increased to 1.02, 2.31, and 2.58 with 6-, 12-, and 18-day algae aging, respectively. The obtained results suggested the vital role of cultivation-medium components in the composition of microalgae metabolites and, consequently, nanoparticle biosynthesis. As previously mentioned, the culture filtrates from the urea and ammonia treatments showed no nanoparticle formation, due to the absence of reductases, while the ammonium sulfate treatment aided the synthesis of relatively small amounts of AgNPs, due to the expected role of sulfate reductase. Extracellular release of reductases enzymes into a surrounding medium is considered to be the main factor for extracellular nanoparticle biosynthesis, especially for AgNPs [55]. Those enzymes are responsible for reduction processes in many microorganisms.