## 4 Discussion
For instance, mixed cultures involving _Chlorella vulgaris_ and _Scenedensmus obliquus_ have shown superior nutrient reduction abilities compared to monocultures [52]. Additionally, employing rotating algae biofilm reactors that include bacteria can increase the SO\({}_{4}\)2- removal efficiency by up to 46% [53]. Research also suggests that DSW can significantly boost nutrient removal, reducing toxicity and enabling more effective management of TN and TP [54]. Incorporating these advanced strategies can significantly enhance the capability of _H. rubescens_ KNUA214, making it a more robust option for sustainable wastewater treatment. During the pigment analysis, the initial results revealed the highest concentrations of astaxanthin, canthaxanthin, and beta-carotene in BG-11 medium on day 4. By day 8, however, the addition of diluted swine wastewater (DSW) significantly enhanced the carotenoid yields, particularly under the BG-11 + 50% DSW condition. In this setup, most carotenoids, except for zeaxanthin, reached their peak concentrations. Specifically, astaxanthin increased 2.71-fold, lutein increased 2.09-fold, canthaxanthin increased 2.56-fold, and beta-carotene doubled compared to the initial levels. These findings suggest that components in the DSW, such as nitrogen and organic matter, acted as stressors that effectively promoted carotenogenesis. Stress-induced carotenoid synthesis is a well-documented phenomenon in microalgae. For instance, _Haematococcus pluvialis_ produces astaxanthin under nutrient deprivation and light stress, primarily through the upregulation of carotenogenic genes such as phytoene synthase (PSY). Nitrogen depletion combined with varying light intensities has been shown to significantly influence astaxanthin accumulation, with optimal synthesis observed under low nitrogen levels and moderate light intensity to minimize cell mortality [55; 56]. Similarly, _Chlorella vulgaris_ demonstrated enhanced carotenoid synthesis, including lutein and beta-carotene synthesis, under nitrogen depletion stress. Stepwise nitrogen depletion has been found to facilitate carotenoid accumulation, while simultaneously affecting photosynthetic efficiency and proliferative activity, emphasizing the role of nitrogen stress as a critical driver of carotenogenesis in this species [57]. These results underscore the pivotal role of stress conditions, such as those induced by DSW, in enhancing carotenoid production. The BG-11 + 50% DSW condition effectively replicated such stressors, offering a promising strategy for maximizing carotenoid yields in _H. rubescens_ KNUA214. This highlights the broader importance of stress physiology in microalgae not only for improving production efficiency but also for advancing sustainable and eco-friendly biotechnological applications. Further research to optimize these conditions is essential for scalable carotenoid bioproduction and sustainable industry practices. The proximate and ultimate analyses provided key insights into the composition and energy content of the microalgal biomass, affirming its potential as a bioenergy feedstock. As shown in Table 1, the uniform moisture content across all the samples indicated that the drying methods after harvest were effective. The high levels of volatile matter suggested that the biomass largely consisted of compounds advantageous for combustion, enhancing burning efficiency [58; 59]. The variations in the ash content, reflecting differences in mineral uptake by the algae, indicated that lower ash values, which minimize slagging and fouling during combustion, make these microalgal strains suitable for bioenergy applications without the need for extensive ash removal [60; 61] 1 C and O levels, typical for organic biomasses, which support its conversion into bioenergy through biochemical or thermochemical processes. The favorable H:C ratios indicated potential for high energy yields [62], while the low S content across the samples implied fewer sulfur oxide emissions, aiding in compliance with environmental standards [63; 64].