*5.2. Astaxanthin*

Commercial production of astaxanthin by *Haematococcus* sp. has been implemented by more than one microalga company (e.g., Cyanotech and Aquasearch); they resorted to a two-stage system, consisting of a first step to  produce green biomass under optimal growth conditions ("green" stage), followed by a second stage when the microalga is exposed to adverse environmental conditions to induce accumulation of astaxanthin ("red" stage) [50]. Astaxanthin productivities in large scale facilities are typically *ca*. 2.2 mg Lƺ1 [39]—even though maximum astaxanthin productivities of 11.5 mg Lƺ1 dƺ1 can be attainedat bench scale [51]. 

Micro Gaia, a marine biotech firm engaged in production of microalgae rich in astaxanthin, proposed a single-step, continuous manufacture process using moderate nitrogen limitation [52,53]: the biomass and astaxanthin productivities obtained were 8.0 and 0.7 mg Lƺ<sup>1</sup> dƺ1, respectively [54]. The feasibility of the latter approach for production of astaxanthin by *H. pluvialis* was tested continuous-wise in outdoor apparatuses [48]: Aquasearch Growth Modules (AGM)—*i.e*., 25,000 L enclosed, computerized photobioreactors, were combined up to three units to obtain large amounts of clean, fast growing *H. pluvialis*; they were transferred daily to a pond culture system, where carotenogenesis and astaxanthin accumulation were induced. After 5 days of synthesis, cells were harvested by gravitational settling— with a typical content of 2.5% (w/wDW) astaxanthin; a high pressure homogenizer was used to disrupt the cells, and then drying was carried out to less than 5% (w/w) moisture. The performance of AGM could be improved 2-fold within the first 9 mo of operation; and the biomass concentration increased from 50 to 90 g mƺ2, with associated productivities increasing from 9 to 13 g mƺ2 dƺ1 within the same period [39]. 

However, the production capacity of *H. pluvialis* was constrained by its intrinsic slow growth, low cell yield, ease of contamination by bacteria and protozoa, and susceptibility to adverse weather conditions [5]. Moreover, *H. pluvialis* cannot be efficiently cultivated in dark heterotrophic mode—so production of astaxanthin should adopt the photosynthetic mode, and thus resort to levels of irradiance (e.g., 950 μmol mƺ2 sƺ1) well beyond what would be economically reasonable [39]. Owing to its ease of culturing and high tolerance to environmental fluctuations, *C. zofingiensis* (another green microalga) has been put forward as an alternative for astaxanthin production: it grows quite fast (*ca*. three times faster than *H. pluvialis*), and accumulates significant amounts of secondary carotenoids in the dark, thus facilitating large-scale cultivation of denser biomass [47,55]. 

Oxidative stress induced by intense illumination has been found to play a crucial role upon astaxanthin synthesis [56]; active oxygen molecules, generated by excess photooxidation caused by high light irradiance, do apparently trigger synthesis of carotenoids as part of a cellular strategy aimed at cell protection against oxidative damage [47]. In particular, flashing light increased the rate of astaxanthin production per photon in *H. pluvialis* by at least 4-fold relative to that under continuous light sources [57]—thus proving that light quality is more important than quantity [58]. 

The effect of irradiance depends also on such operating variables as culture density, cell maturity (flagellates are much more sensitive than palmelloids), medium nutrient profile and light path[59]. The predominant role of light stress and nitrogen deprivation towards induction and enhancement of biosynthesis in the aplanospores of *H. pluvialis* was originally suggested in the 1950s [60]; astaxanthin accumulation comes along with growth halting, as happens in most cases of stress imposed upon microalgae [59,61]. Imamoglu *et al.* [54] compared the effect of various stress media, under high light intensities, upon astaxanthin accumulation; those authors concluded that addition of CO2 in an N-free medium, under 546 μmolphoton mƺ<sup>2</sup> sƺ1, were the best conditions for accumulation of astaxanthin—which attained *ca*. 30 mg gƺ1. 

Astaxanthin may thus be efficiently produced outdoors in continuous mode, if accurate nitrate dosage is provided [48]; besides N, such oligoelements as iron play a role. This essential oligoelement takes part in assimilation of nitrate and nitrite, deoxidation of sulphate, fixation of N, and synthesis of chlorophyll [62–65]. Iron deficiency was reported to constrain microalga growth, even in rich nutrient media [64]; whereas its addition enhanced astaxanthin synthesis [66–69]. Cai *et al*. [67] further tested how iron electrovalencies and counter ions affect cell growth and accumulation of astaxanthin; 18 μmol Lƺ1 Fe2+-EDTA stimulated synthesis of astaxanthin more effectively, up to contents of 30.7 mg gƺ1; and despite the lower cell density attained (2.3 × 105 cell mLƺ1), a higher concentration (36 μmol Lƺ1) of FeC6H5O7 yielded cell density and astaxanthin production levels that were 2- and 7-fold those reached under iron-limitation. 

In the "red stage" of growth, *Haematococcus* cells require only carbon as major nutrient—which this is usually supplied via directly injecting CO2 into the photobioreactor during daylight [61]. Furthermore, high irradiance provides more energy for photosynthetic fixation of C, which leads to a higher rate of astaxanthin synthesis [68];this may be further enhanced by raising the C/N ratio [69]. 

Finally, Chen *et al.* [70] experimented with heterotrophic conditions—using pyruvate, citrate and malate as substrates, towards synthesis of astaxanthin by *C. zofingiensis* in the absence of light. Presence of any of the aforementioned substrates above 10 mM stimulated biosynthesis of astaxanthin (and other secondary carotenoids); *ca*. 100 mM pyruvate led to yields of 8.4–10.7 mg Lƺ1 astaxanthin, which correspond to a 28%-increase. 

## *5.3. Ά-Carotene*

Semicontinuous cultivation of *D. salina* at 25 °C produced 80 g mƺ3 dƺ1 biomass [42]—from which 1.25 mg Lƺ1 of Ά-carotene was recovered [71]; however, this figure could be improved up to 2.45 mg mƺ3 dƺ1 in continuous biphasic bioreactors [72]. When cultivated photoheterotrophically, a significant increase of cellular Ά-carotene  content was experimentally observed: the maximum score was 70 pg cellƺ1, in a culture enriched with 67.5 mM acetate and 450 μM FeSO4 [33]. 

As with astaxanthin, Fe2+ plays an important role in Ά-carotene accumulation in *D. salina*; by inducing oxidative stress, those cations stimulate said synthesis, especially in the presence of a carbon source. UV-A radiation (320–400 nm) added to the photosynthetically active radiation (PAR, *i.e*., 400–700 nm) can be regarded as another stress factor during growth of, and carotenoid accumulation by *Dunalliela bardawil;* compared with cultures exposed to PAR only, addition of 8.7 W mƺ2 UV-A radiation to 250 W mƺ2 PAR stimulated long-term growth of that microalga, and led to a 2-fold enhancement in Ά-carotene accumulation by 24 d [38]. 

## **6. Extraction and Purification**

Although microalga-mediated synthesis of carotenoids is crucial in biotechnological production thereof, a major portion (if not most) of their cost actually lies on downstream separation—e.g., biomass drying and disruption, followed by solvent extraction and purification. Hence, these issues are addressed below, in view of their importance toward commercial scale processes. 
