*2.2. Carotenoid accumulation and xanthophylls cycle activity in urea grown* Coccomyxa acidophila *cells*

*Coccomyxa acidophila* accumulates commercial value carotenoids including lutein, β-carotene and zeaxanthin (Figure 4). Besides assessment of the best nitrogen sources for biomass production, carotenoid accumulation in urea and nitrate grown cultures was also studied (Figure 5). According to the best growth conditions inferred from Figure 1, for this experiment, the carotenoid content was followed in urea grown cultures (fluidized with air) and in nitrate grown cultures (fluidized with air supplemented with 5% v/v CO2). In addition, carotenoid content was also followed in nitrogen-deprived cultures, as nitrogen depletion is a very well known carotenogenic condition for many microalgae species. In good agreement with the enhanced cell growth in urea grown cultures, total carotenoid content in the reactor also increased much more rapidly in urea grown cultures than in control cultures. Consequently, the carotenoid content of urea grown cultures ( g·mL 1) became about two-fold that of the nitrate grown cultures (control cultures), until late  exponential growth phase (Figure 5). This could be due to the increased biomass production in urea grown batch cultures, therefore higher carotenoid content in the reactor is not necessarily a consequence of faster carotenoid biosynthesis. However, simple calculations of the content of specific carotenoids per cell revealed a prompt carotenoid biosynthesis enhancement (namely -carotene, lutein, zeaxanthin) in urea grown *Coccomyxa acidophila* cells, as can be inferred from the carotenoid cell content data in Figure 6; lutein by far being the most abundant carotenoid. This means that urea clearly promotes increases in lutein and β-carotene cell content, at least up to late exponential growth phase, where lack of nutrients, shading effect and stress factors change the observed trend. Besides, it can be observed that cell content of violaxanthin inversely correlates with cell content of zeaxanthin over the time course. This is the first evidence of an active xanthophylls cycle in *Coccomyxa acidophila* that converts violaxanthin into zeaxanthin by means of violaxanthin deepoxidase activity. Interestingly, nitrogen starvation did not promote carotenoid accumulation in *Coccomyxa acidophila* cultures, unlike other common microalgae including *Dunaliella*, *Haematococcus* and many others [23,24]. 

**Figure 4.** HPLC chromatogram showing the main carotenoids of *Coccomyxa acidophila*. AU: arbitrary units. 

**Figure 5.** Total carotenoid content in *Coccomyxa acidophila* cultures grown on different nitrogen sources or under nitrogen starvation. Air alone or CO2 in air (5% v/v) were used as carbon source, as indicated for each culture within the Figure legend. 

**Figure 6.** Time-course of the cell content of the indicated specific carotenoids in *Coccomyxa acidophila* cultures incubated in either nitrate or urea. 

Fernández-Sevilla *et al.* [25] recently reviewed the biotechnological production of lutein. The paper includes an updated list of lutein production experiments performed on different scales using microalgae species. Table 2 shows the most relevant data of lutein productivity by microalgae and reactor type used for the production processes. Considering intracellular lutein cell content of each one of the promising species and lutein productivity in photobioreactors, *Scenedesmus almeriensis* [26], *Muriellopsis sp.* [27] and *Chlorella protothecoides* [28] emerge so far as the most efficient strains for the biotechnological production of lutein from microalgae. When incubated under standard culture conditions, *Coccomyxa acidophila* onubensis accumulates up to 6.1 mg·g 1 dry weight, which is within the upper range of lutein concentrations accumulated by the above mentioned microalgae. We are now running continuous cultures of *C. acidophila* in tubular laboratory photobioreactors in order to obtain lutein productivity data in long-term (weeks) production processes. Compared to continuous cultivation of other lutein producing species, *C. acidophila* has the practical advantage of growing well in an extremely selective culture medium at very low pH which preserves cultures from microbial contamination. 


**Table 2.** Lutein productivity of lutein-enriched microalgae. 
