## 3 Results and Discussion
### Sequential Batch Photobioreactor Experimentation with Duckweed and microalgae
#### 3.3.3 Microalgae bioremediation in Duckweed-Treated RW Media  
The three supernatants obtained from PBR-1 (TRW 10% to 30%) served as culture media in the PBR-2; consequently, the initial concentrations of each of the pollutants in PBR-2 are the same as the final concentrations in PBR-1. Figure 7 shows the temporal changes in the concentrations of (A) COD, (B) nitrates, (C) phosphate, and (D) sulfate in the supernatants for both _Chlorella_ and _Scenedesmus_ cultures. At first sight, both microalgae significantly reduced the concentration of the four pollutants in each of the three culture media over seven days of cultivation in PBR-2. The reduction rate was highest for pollutants initially present at higher concentrations in the TRW% medium. Nitrates were completely depleted in all TRW% for both microalgae, whereas phosphates and sulfates were only depleted in TRW-10% with _Scenedesmus_. As the consumption kinetics of Figure 7 suggested that both MEDIUM and ALGA factors could influence the pollutant removal efficiency (pollutant-RE%), a multivariate analysis of variance (MANOVA) was carried out, and results are represented in Figure S4 (Supplementary Material). It revealed a significant main effect of the MEDIUM factor on the combined pollutant-RE%, Wilk's Lambda = 0.018, F (8, 18) = 14.74, \(p\) < 0.001 (Figure S4A,D,G,J). Subsequent univariate tests showed that MEDIUM significantly influenced three pollutant-RE%: COD-RE% (F = 136.5, \(p\) < 0.001), NO3\({}^{-}\)-RE% (F = 17.7, \(p\) < 0.001), and PO4\({}^{3-}\)-RE% (F = 5.4, \(p\) < 0.021). This observation confirms the importance of adjusting TRW% to effectively modulate pollutant removal rates, offering a means to enhance bioremediation outcomes by tailoring medium composition to specific environmental conditions. Figure 6: Comparison of the effect of varying proportions of raw whey (RW) pretreated with duckweed in the 4 L photobioreactor (TRW%) in culture media versus without pretreatment (RW%) on the growth kinetics of _Chlorella_ sp. MC18 (CH) and _Scenedesmus_ sp. MJ23-R (SC) grown in flask in batch mode. (**A,B**) Natural logarithm of the normalized cell concentration (\(X/X_{0}\)) versus culture time. (**C**) Photograph showing part of the microalgae flask cultures with TRW% carried out in the cultivation system used. (**D–L**) Evaluation from a two-way ANOVA of the main effects MEDIUM  
and ALGA and their interaction (MEDIUM-ALGA) on the growth kinetic parameters mean \(\mu_{max}\) (**D**-**F**), \(P_{B}\) (**G**-**I**), and \(Y_{B}\) (**J**-**L**). Each MEDIUM (**D**,**G**,**J**) and ALGA group (**E**,**H**,**K**) gathers all the results obtained for the two microalgae and for the seven MEDIUM levels, respectively. Bars around points represent 95.0% confidence intervals. Overlapping bars indicate no significant difference. Similarly, there was a significant main effect of the ALGA factor on the combined pollutant-RE%, Wilk's Lambda = 0.015, F (4, 9) = 147.15, \(p\) < 0.001 (Figure S4B,E,H,K), particularly affecting COD-RE% (F = 251.3, \(p\) < 0.001), PO43\({}^{-}\)-RE% (F = 15.5, \(p\) < 0.002), and SO4\({}^{2-}\)-RE% (F = 136.0, \(p\) < 0.001), but not in NO3\({}^{-}\)-RE%. _Chlorella_ or _Scenedesmus_ exhibit varying efficiencies in pollutant removal, emphasizing the need for careful consideration when choosing species for bioremediation applications. By selecting microalgae with optimal pollutant removal capabilities, researchers can maximize remediation efficiency and improve overall treatment outcomes. Figure 7: Bioremediation effectiveness of treated raw whey influent (TRW) pollutants by _Chlorella_ sp. MC18 (CH) and _Scenedesmus_ sp. MJ23-R (SC) cultures in the 4 L photobioreactor (PBR-2) at the TRW% proportions of 10%, 20%, and 30% in the culture medium. Pollutant disappearance kinetics in the culture: (**A**) COD; (**B**) nitrate, NO3\({}^{-}\); (**C**) phosphate, PO4\({}^{3-}\); and (**D**) sulfate, SO4\({}^{2-}\). (**E**) Photograph showing part of the microalga PBR-2 cultures carried out in the cultivation system used.