## 3 Results and Discussion
### Sequential Batch Photobioreactor Experimentation with Duckweed and microalgae
#### 3.3.1 Bioremediation Potential of Duckweed in RW-Based Media  
PBR-1 pretreated the culture media RW-10%, RW-20%, and RW-30% at the three initial duckweed biomass surface densities (DEN) of 2.50, 3.75, and 5.00 g dm\({}^{-2}\). To facilitate comparison, the percentage production of duckweed biomass (\(Y_{PBR-1}\)) was calculated relative to DEN in each PBR-1. The results of a two-way ANOVA for the factors RW% and DEN are depicted in Figure 4. Both RW% and DEN showed statistically significant effects (\(p<0.001\)) on \(Y_{PBR-1}\) (F (2, 18) = 1801, F (2, 18) = 269, respectively). Although the contribution of the RW%-DEN interaction to the variance was also significant (\(p<0.001\); F (4, 18) = 60), it was much lower than that of the main factors. In PBR-1, biomass increased when RW-10% was used but declined with higher RW% (Figure 4A). In contrast, \(Y_{PBR-1}\) increased with DEN (Figure 4B). Notably, the decline in \(Y_{PBR-1}\) at 5.00 g dm\({}^{-2}\) DEN from RW-10% to RW-30% was less pronounced compared to 2.50 and 3.75 g dm\({}^{-2}\), respectively (Figure 4C). The above trends were consistent with those observed for \(Chl_{total}\) in flask cultures (Figure 2), suggesting that \(Chl_{total}\) served as a proxy for \(Y_{PBR-1}\). Additionally, as shown in Figure 4C, the initial surface density of 5.00 g dm\({}^{-2}\) yielded the highest \(Y_{PBR-1}\) values across all RW% conditions, with the maximum value (22.9% \(\pm\) 5.5%) attained at RW-10%, corresponding to a productivity of 0.194 \(\pm\) 0.053 g dm\({}^{-2}\) d\({}^{-1}\). Therefore, the effluent from PBR-1 was used as influent in PBR-2 for further treatment with microalgae at this surface density. The bioremediation effectiveness of duckweed at RW-10%, RW-20%, and RW-30% in PBR-1 is depicted in Figure 5 for an initial inoculum surface density of 5.00 g dm\({}^{-2}\). Observably, COD, nitrates, phosphate, and sulfate concentrations decreased in all three culture media, with the most significant reduction observed for nitrates, which were completely depleted in RW-10% and RW-20%. A one-way MANOVA test was conducted to assess the impact of RW% on removal efficiencies (RE%) of the four pollutants (Figure 5E). Multivariate tests revealed a highly significant effect of RW% on the combined RE% values (Wilk's Lambda = 0.001, F (8, 6) = 25.9, \(p<0.001\)), indicating an overall influence. Univariate tests further demonstrated significant effects of RW% on RE%-COD (F (2, 6) = 28.4, \(p<0.001\)), RE%-nitrate (F (2, 6) = 31.5, \(p<0.001\)), RE%-phosphate (F (2, 6) = 29.3, \(p<0.001\)), and RE%-sulfate (F (2, 6) = 7.9, \(p<0.021\)). These findings underscore the influential role of RW% in shaping duckweed performance in RE%. According to Figure 5A, COD removal was notably high and unexpected, probably due to several factors. First, during the treatments with duckweed at each RW concentration (10%, 20%, or 30%, \(v/v\)), there was significant removal of TSS, particularly a cream or fine white particles present in each %RW (see Supplementary Material). This cream adhered to the roots and internal surfaces of each macrophtye seedling. Whey cream (WC), a component of cheese whey, consists mainly of total solids, lactose, protein, fat, phospholipids, and total nitrogen [48,49], all of which contribute to the organic matter in %RW media. A recent study by [50] suggests that the roots of duckweed act as a filter, trapping suspended particles in the water, which reduces high levels of TSS and COD when _Lemma minor_ is grown in dairy effluent. From a resource recovery perspective, traditionally expensive methods (e.g., centrifugal force) are needed to separate whey cream from cheese whey [49]. However, cultivating _Lemma minor_ in PBR-1 not only remedies RW but also allows the recovery of WC and macrophtye biomass, both of which are high-value bioproducts worth exploring in the future.