## 1 Introduction  
Alternative sources of nutrients and food ingredients rich in bioactive compounds have sparked great interest in the last years, particularly due to their benefits for human and animal health [1; 2]. Microalgae can produce a wide range of metabolites such as proteins, lipids, carbohydrates, and valuable pigments with relevant bioactivities that can be used to enhance the nutritional value of food and feed, providing significant health benefits [3; 4; 5]. Furthermore, these micro-organisms have the great advantage of growing in industrial reactors, preventing their competition with food crops for fresh water supply and arable land. In addition, they can be harvested daily and offer higher reliability and even seasonal-independence, particularly if they are grown under controlled conditions indoors, as in heterotropy [6; 7; 8]. Finally, microalgae industrial production still entails high production costs, and the scale-up of cultures from the laboratory scale to industrial photobioreactors is a highly time-consuming process that needs to be addressed [9].  
Besides photoautotrophic growth, some microalgae species have the ability to grow heterotrophically using alternative metabolic pathways [10; 11]. Heterotrophic cultivation implies the ability to grow in the absence of light, with organic carbon supplementation to replace light energy [12]. This type of cultivation appears to be an alternative to more economical industrial cultivation since it enables stricter control of the process parameters and higher growth rates, leading to cell concentrations in the order of 100 g\(\cdot\)L\({}^{-1}\)[11; 12; 13; 14] and a reduction in harvesting costs [15]. Nevertheless, this cultivation mode decreases the protein and pigment contents in the resulting biomass [15]. According to _[PERSON]_ et al. [9], a two-stage approach, combining heterotrophic inoculum and photoautotrophic cultivation, might be an advantageous strategy to improve industrial production since it overcomes these limitations and provides high-quality biomass.  
Moreover, increasing microalgae growth rates and biomass productivity while reducing operational costs is essential for the future of industrial producers. In addition, the production of different microalgal species is key to promoting a thorough portfolio of bioactive compounds. These compounds can be of interest for different biotechnological applications towards the development of sustainable and economical solutions for today's needs [16; 17]. In addition, the appropriate culture medium recipes, a factor that is crucial for obtaining highly concentrated microalgal cultures, must be developed to enhance biomass and target metabolite productivities [18].  
In order to achieve the aforementioned goal, the use and design of experiment methodologies has been applied, which allows for a reduction in time and the number of experiments and is more accurate than 'one variable at a time methods' since it evaluates interactions between the variables tested [19; 20]. Response surface methodology (RSM) is a compilation of mathematical/statistical techniques that allow for the designing of experiments and the assessment of their effects and significance, and the monitoring of the interaction of several variables in specific responses through the evaluation of empirical models [21]. RSM has become a fundamental tool in numerous branches of scientific and technological research, such as biotechnology, namely in medium-optimization studies [22; 23]. Therefore, the present work focused on the optimization of photoautotrophic and heterotrophic culture media for a novel microalga, previously known as _Oophila amblystomatis_ and recently reclassified as _Chlorococcus amblystomatis_[24]. The biomass composition of _C. amblystomatis_ was evaluated regarding protein, pigments, lipid, and carbohydrate contents to determine the nutritional profile attained in each cultivation condition.