## 4 Discussion  
The light source and wavelength played important roles in improving astaxanthin and biomass yield during the cultivation of _H. pluvialis_. Although various colours of light are used for microalgal cultivation, mainly red light (600-700 nm) and blue light (400-500 nm) are utilized [43]. The red and blue wavelengths of visible light assist in cell division at the vegetative stage and cell maturation at the astaxanthin accumulation stage, respectively. Blue light is more important, as it passes through clear water and regulates metabolic pathways. Astaxanthin accumulation and dry biomass were higher when red and blue light-emitting diodes (LEDs) were used together, compared to their separate use at an intensity of 100-120 umol m\({}^{-2}\) s\({}^{-1}\). A comparative study of monochromatic red and mixed red/blue light similarly showed that red/blue light at a 2:1 ratio produced higher biomass [44; 45; 46]. The use of blue LEDs (440 nm) enhanced the biosynthesis of astaxanthin in _H. pluvialis_[47].  
This study examined the growth of _H. pluvialis_ cultures in the green and red phases separately. In the green stage, inorganic nutrients were provided for growth; as low light intensity was preferred, the light intensity was fixed at 25 umol m\({}^{-2}\) s\({}^{-1}\). Microalgae generally stop dividing at light intensity levels below 60 umol m\({}^{-2}\) s\({}^{-1}\)[48], whereas _Haematococcus_ cells divided at lower levels in this study. High light intensity triggers the formation of astaxanthin in microalgae. Lighting frequency, duration, intensity, and spectral composition direct the growth and biosynthesis of compounds in microalgae [32]. Astaxanthin accumulation in _Haematococcus_ requires cultivation under stress conditions such as nutrient deficiency or exposure to high light intensity. As nitrogen depletion is also involved in the induction process, photoinhibition is highly relevant as it depends on the de novo synthesis of the D1 protein [49].  
However, our results also suggested that these light intensity effects have an upper limit. Biomass and carotenoid contents were approximately 15% higher at a light intensity of 160 umol m\({}^{-2}\) s\({}^{-1}\) than at 220 umol m\({}^{-2}\) s\({}^{-1}\). A previous study reported that growth and astaxanthin production were increased at 170 umol m\({}^{-2}\) s\({}^{-1}\)[50]; in another, an organic carbon source was added open culture was performed under light intensity conditions of 150 and 300 umol m\({}^{-2}\) s\({}^{-1}\)[51]. Bacterial contamination is inevitable in outdoor microalgal culture [52], and the risk increases when organic carbon is added to the medium [53]. However, the number of bacterial cells in water has been shown to be a function of phosphorus and nitrogen concentrations [54]. Bacterial growth is more accurately described by its high affinity for nutrients that are readily available in the culture medium [55]. Therefore, astaxanthin accumulation in _H. pluvialis_ under nitrogen depletion conditions should be studied further. Interestingly, the values of Chl \(a\) and Chl \(b\) were found to be similar in this study. According to [56], red light (600-700 nm) and blue light (420-450 nm) are absorbed by chlorophyll. Therefore, the mechanism behind this needs to be further explored in future research.