## 3 Results and Discussion
### Effect of Light Intensity on Carotenoids and Astaxanthin Production by H. pluvialis  
Figure 2 illustrates the impact of different light regimes on the total carotenoids and astaxanthin content in _H. pluvialis_ cultivated mixotropbycally in BWW. At a light intensity of 35 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\), the BWW-BG11 1:1 medium yielded the highest astaxanthin and total carotenoids content (2.32 \(\%\)dw and 28.9 mg g\({}^{-1}\)dw, respectively), outperforming the control BG11 (2.15 \(\%\)dw and 26.8 mg g\({}^{-1}\)dw) (Figure 2a). When exposed to higher light intensity (Figure 2b), the BWW medium achieved superior results (2.47 \(\%\)dw and 30.8 mg g\({}^{-1}\)dw) compared to the control (1.50 \(\%\)dw and 17.8 mg g\({}^{-1}\)dw) in both total carotenoids and astaxanthin content. Figure 2c shows that under a light shift, the BWW-BG11 1:1 medium produced the highest astaxanthin content (2.12 \(\%\)dw), followed closely by BWW (1.98 \(\%\)dw), both slightly surpassing the control (1.90 \(\%\)dw). Also, in terms of total carotenoids content, BWW-BG11 1:1 outperformed BG11, with values of 26.4 mg g\({}^{-1}\)dw and 23.6 mg g\({}^{-1}\)dw, respectively. _H. pluvialis_ is characterized by a complex life cycle and slow growth rates, making large-scale cultivation challenging. However, using organic carbon sources in its cultivation can enhance growth rates and promote the inductive stage, which leads to astaxanthin accumulation [39]. In mixotrophic cultures, organic carbon sources are known to stimulate cell division, hematocyst formation, and carotenoid accumulation [40]. BWW composition involves volatile fatty acids, sugars, soluble starch, celluloses, and alcohols, capable of sustaining mixotrophic metabolism. Additionally, since astaxanthin is widely used in the food and feed industries, the growth medium for _H. pluvialis_ must be free for toxic chemicals [41]. This is crucial when selecting appropriate WW streams for cultivating microalgae for astaxanthin production. It is hypothesized that WW from the food industry, which typically avoids toxic chemicals, would be ideal for this purpose. The literature provides evidence on the feasibility of using various types of WWs, including agro-industry waste, to extract astaxanthin from _H. pluvialis_[42; 43]. Based on these considerations, BWW was selected for this study as a source of organic carbon to facilitate the transition of _H. pluvialis_ from the vegetative stage to the inductive one. Figure 2: Total carotenoids (mg \(g^{-1}\)dw) and astaxanthin content (%dw) of _H. pluvialis_ grown under mixotrophy in 4 PBRs systems (BWW, BWW-BG11 1:1, BWW-BG11 1:3, BG11) using three different light regimes: (**a**) 35 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\), (**b**) 140 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\), (**c**) 35 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) and 140 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\). Mean differences were compared using 1-way ANOVA with Dunnet correction (n = 6, * \(p\) < 0.05 (Unmarked); **_p_ < 0.001; *** \(p\) < 0.0001). The color transition of _H. pluvialis_ from the green to red stage under a dual light shift from 35 to 140 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\) is depicted in Figure 3. Figure 3a shows the initial conditions on day 0, where _Haematococcus_ was inoculated in three mixotrophic systems: BWW (PBRs 1a and 1b), BWW-BG11 1:1 (PBRs 2a and 2b), BWW-BG11 1:3 (PBRs 3a and 3b), and in the control BG11 (PBRs 4a and 4b). By the fifth day, when the light intensity was increased from 35 to 140 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\), the PBRs 1a and 1b (BWW) clearly exhibited a reddening stage, while the other three systems displayed varying shades of green, with the BWW-BG11 1:3 system showing a strong green color (Figure 3b). By the 10 th day, after _Haematococcus_ had been exposed to the higher light intensity for five days, the PBRs 1a and 1b (BWW) confirmed their red stage, while PBRs 2, 3, and 4 had turned dark, with little green remaining (Figure 3c).