## 3 Results and Discussion
### Effect of Light Intensity on Carotenoids and Astaxanthin Production by H. pluvialis
The color changes observed in our study are consistent with those reported by [17], where their system 0 N corresponds to BWW in our study, and the other three systems with increasing N concentration (0.5-3 N) can be compared to our BWW-BG11 mixtures and the control. The images in Figure 3 suggest that _H. pluvialis_ accumulated more biomass (in terms of chlorophyll \(a\) and _b_) in the control and in the BWW-BG11 mixtures, while the early reddening stage in the BWW system indicates an initial focus on astaxanthin accumulation rather than chlorophylls. This observation is supported by the chlorophylls, carotenoids, and carotenoid/chlorophyll ratio data presented in Table 4, which cover all PBRs under the three light regimes. By the 10 th day of Exp. 3, TChI accumulation in BWW was very low (0.50 mg L\({}^{-1}\)) compared to the other three systems, BWW-BG11 1:1, BWW-BG11 1:3, and BG11, which exhibited much higher total chlorophylls levels (3.14 mg L\({}^{-1}\), 3.39 mg L\({}^{-1}\), and 1.36 mg L\({}^{-1}\), respectively). The lowest chlorophyll \(a\) and \(b\) levels, as well as total chlorophyll content across all three light regimes, were observed when _Haematococcus_ was exposed to 140 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\) from the start (Exp. 2). Under these conditions, _Haematococcus_ cultivated in BWW had the highest total carotenoid accumulation (0.79 mg L\({}^{-1}\)) and astaxanthin content (2.47%) (Table 4 and Figure 2b). Increased light intensity enhances the photosynthetic machinery, leading to higher reactive oxygen species (ROS) production. To mitigate the oxidative stress caused by ROS, _Haematococcus_ redirects its metabolic pathways towards the synthesis of astaxanthin, which effectively neutralizes the ROS and stabilizes cellular components. However, while high light intensity can significantly boost astaxanthin production, it must be carefully balanced to prevent excessive photo-damage or growth inhibition. Optimal light conditions typically involve a balance between high light exposure and other factors like temperature, nutrient availability, and light duration to maximize both biomass and astaxanthin yield [44]. Figure 3: Astaxanthin accumulation in the 4 PBRs systems (BWW 1a and 1b, BWW-BG11 1:1 2a and 2b, BWW-BG11 1:3 3a and 3b, BG11 4a and 4b) at day 0 (**a**), day 5 (**b**) and day 10 (**c**) with a light intensity of 35 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\)during the first 5 days and 140 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\) during the remaining 5 days. The morphology of \(H\). _pluvialis_ before and after cultivation was examined using a digital microscope and is shown in Figure 4. Day 0 represents the initial culture conditions with low light (35 umol m\({}^{-2}\) s\({}^{-1}\)) across all four PBR systems, while day 10 depicts the cells after five days of exposure to high light (140 umol m\({}^{-2}\) s\({}^{-1}\)). Initially, the cells were light green, indicating chlorophyll presence. As cultivation progressed, young \(H\). _pluvialis_ cells lacked thick cell walls, which became more robust during the green stage (images not shown). The increased thickness of the layered cell wall is a protective strategy during astaxanthin accumulation [45]. As the cells began to accumulate astaxanthin, their color transitioned to reddish green, signaling that the culture is ready to enter to the maturation stage, culminating in full astaxanthin accumulation by day 10, as shown in Figure 4.