## 1 Introduction
The vast majority of studies and industrial application of _H. pluvialis_ cultivation have employed NaNO\({}_{3}\) as a nitrogen source. NaNO\({}_{3}\), however, is expensive and nitrogen only accounts for 16.5% of the molecular mass of the chemical, against 46% in urea containing two nitrogen atoms. For this reason, urea would be required in lower quantities for an equivalent nitrogen concentration. Attempts to cultivate _H. pluvialis_ in urea commonly fail and _H. pluvialis_ mostly used growth media, and does not provide urea in their formulation [15; 16; 17]. Deeper investigations and manipulations of the abiotic conditions could establish the use of urea in _H. pluvialis_ growth mediums for cost-effective large-scale production. The second phase of the two-step cultivation process is the carotenogenesis induction and can be achieved by modulating a few growth parameters: light and N depletion are commonly employed as stressors, and iron addition and osmotic stress may also enhance the accumulation of astaxanthin [18]. The astaxanthin accumulation employing autotrophic growth, however, may not have reached its maximum potential [15; 16]. Currently, 4% \(w/v\) of astaxanthin accumulation in _H. pluvialis_ is often considered the common threshold for comparative success [19; 20]. The main difficulty in applying a stress to the _H. pluvialis_ cells is to find the threshold level which minimises biomass loss and cell bleaching and maximises astaxanthin accumulation. It has been ascertained that high levels of stress, such as a sudden increase in irradiance or nitrogen starvation, may damage cells, decreasing the final biomass yield and the amount of recoverable astaxanthin [21]. For example, high light intensity can lead to photoinhibition [22], while nitrogen depletion affects photosynthesis and cellular metabolism [23; 24; 25]. In this study, _H. pluvialis_ (CCAP 34/1D) was selected for a two-step cultivation based upon a previously published strain screening by [PERSON] [26]. In our study, an initial optimisation of the growth conditions was carried out for both the green and red phases of the strain CCAP 34/1D: nutrients concentration and pH were manipulated as shown in Supplementary Materials 1. Afterward, different NS were tested to enhance biomass productivity during the green phase of growth. To induce the red phase, the drawbacks of conventionally applied stringent individual stressors that were mentioned above were considered. We hypothesised that multiple moderate stressors could be simultaneously applied for a possible synergistic effect whilst preserving the biomass accumulated in the green phase. Based on previous works, a mix of stressors (e.g., combining higher irradiance, salinity, organic carbon sources) were tested (Supplementary Materials 2); the mix of stressors that allowed the highest biomass yield preservation coupled with the greatest accumulation of carotenoids was selected to induce the red phase, i.e., buffered JM medium (without the addition of NaNO\({}_{3}\), but retaining the NS from Ca(NO\({}_{3}\))\({}_{2}\cdot\)4H\({}_{2}\)O and (NH\({}_{4}\))6 Mo\({}_{7}\)O\({}_{24}\cdot\)4H\({}_{2}\)O modified with the addition of 0.25% (\(w/v\)) sodium acetate and exposed to 250 umol photons m\({}^{-2}\) s\({}^{-1}\) for 7 days. The dual purpose of this study is to find a simple low-cost strategy to increase the rate of vegetative growth and biomass accumulation, followed by an enhancement of astaxanthin accumulation without any deleterious effects upon biomass yield. The efficiency of the employed induction method and the pigments' characterisation was assessed by HPLC analysis in the green and the red phases. The overarching goal was to find an economic and easily implementable manner to cultivate _H. pluvialis_ CCAP 34/1D for astaxanthin production.