Manufacture of carotenoids via microbiological routes has undergone a greater and greater scientific and commercial importance within the alimentary and aquaculture fields [15], especially in view of environmental and health awareness by consumers at large.

Recall that most oxidation reactions in foods are deleterious—e.g., degradation of vitamins, pigments and lipids, with consequent loss of nutritional value and development of off-flavors [16,17]. Antioxidants—which are adventitious in, or deliberately added to foods, can inhibit oxidation or slow down initiation by free alkyl radicals, as well as interrupt propagation of such free radical chains. The threshold of synthetic food additives legally permitted has been steadily decreasing, due to their suspected role as promoters of carcinogenesis, besides claims of liver and renal toxicities [18]; hence, substitution thereof by natural pigments has become common practice. One good example is the application of Dunaliella spp. for mass production of carotenoids aimed at a preservation role [19,20]. Another advantage of carotenoids is that they are not affected by the presence of ascorbic acid, often used as acidulant to constrain unwanted microbial growth, nor by heating/freezing cycles employed in foods with a similar goal.

On the other hand, carotenoids are particularly strong dyes, even at levels of parts per million. Specifically, canthaxanthin, astaxanthin and lutein from Chlorella have been in regular use as pigments, and have accordingly been included as ingredients of feed for salmonid fish and trout, as well as poultry—to enhance the reddish color of said fish or the yellowish color of egg yolk [4,21–23]. Furthermore, β-carotene has experienced an increasing demand as pro-vitamin A (retinol) in multivitamin preparations; it is usually included in the formulation of healthy foods, although only under antioxidant claims [24–26].

In the human being, oxidation reactions driven by reactive oxygen species can lead to protein damage and DNA decay or mutation; these may in turn lead to several syndromes, viz. cardiovascular diseases, some kinds of cancer and degenerative diseases, and ageing at large [17,27]. As potent biological antioxidants, carotenoids are able to absorb the excitation energy of singlet oxygen radicals into their complex ringed chain—thus promoting energy dissipation, while protecting tissues from chemical damage. They can also delay propagation of such chain reactions as those initiated by degradation of polyunsaturated fatty acids—which are known to dramatically contribute to the decay of lipid membranes, thus seriously hampering cell integrity [21].

One illustrative example is the decline of cognitive ability accompanying Alzheimer’s disease, which is apparently caused by persistent oxidative stress in the brain [28]. Using transgenic mice fed with extracts from Chlorella sp. containing β-carotene and lutein, Nakashima et al. [29] claimed significant prevention of cognitive impairment. Wu et al. [30] used also Chlorella extracts containing 2–4 mg/g_DW of lutein, and reported reduction in the incidence of cancer, as well as prevention of macular degeneration [31]. Likewise, carotenoids extracted specifically from Chlorella ellipsoidea and Chlorella vulgaris inhibited colon cancer development [23]. Furthermore, astaxanthin obtained from Haematococcus pluvialis decreased expression of cyclin D1, but increased that of p53 and some cyclin kinase inhibitors of colon cancer cell lines [32].

Carotenoids have also the ability to stimulate the immune-system, thus being potentially involved in more than 60 life-threatening diseases—including various form of cancer, coronary heart diseases, premature ageing and arthritis [33]; this is specifically the case of canthaxanthin and astaxanthin, and other nonprovitamin A carotenoids from Chlorella but to a lesser degree [23]. A few epidemiological studies encompassing β-carotene from Dunalliela sp.—which contains readily bioavailable 9-cis and all-trans stereoisomers (ca. 40% and 50%, respectively), have indeed provided evidence of a lower incidence of several types of cancer and degenerative diseases [34]. Finally, carotenoids exhibited hyperlipidemic and hypercholesterolemic effects [19].

The most important factors that affect lutein content in microalgae are temperature, irradiance, pH, availability and source of nitrogen, salinity (or ionic strength) and presence of oxidizing substances (or redox potential); however, specific growth rate also plays a crucial role.

High temperature favors accumulation of lutein, as happens with other carotenoids (e.g., β-carotene) in Dunaliella sp. [42], close to the limit of environmental stress; further temperature increases would thus be harmful, and eventually reduce biomass productivity.

A high irradiance level appears beneficial—but its effect depends on whether indoor or outdoor cultivation is considered; in vitro mimicking of all parameters that characterize outdoor operation, e.g., solar cycle and temperature fluctuation, is indeed difficult. Furthermore, the concentration of molecular oxygen outdoors cannot be manipulated, despite its interacting with illumination and temperature. Both irradiance and temperature influence the rate of lutein production, yet cultures of Murielopsis sp. and Scenedesmus almeriensis produced contradictory results; hence, these two factors should be considered in a combined, rather than independent fashion [8].

Likewise, the reported effects of pH are not consistent between batch and continuous cultivations. In the former, lutein content increased at extreme pH values, whereas the best results under continuous operation were observed at the optimum pH for growth rate. It is worth noting that pH is particularly relevant in microalgal cultures because it interferes with CO₂ availability (which is essential for photosynthesis); hence, continuous supply of CO₂, as a fraction of the aeration stream, and pH-controlled injection lead to different results. In general, the maximum lutein productivity is achieved at the optimum pH for biomass productivity [45].

The concentration of nitrogen in the culture medium (in the form of nitrate) does not apparently cause a significant effect upon the lutein content of biomass; however, n-limitation reduces biomass productivity, and consequently leads to poor overall lutein synthesis. Hence, nitrate should be supplied to a moderate excess—so that growth rate is not hampered, while avoiding saline stress that dramatically affects culture performance [8].

Lutein synthesis is enhanced via addition of such chemicals as H₂O₂ and NaClO, which behave as inducers: in the presence of Fe²⁺, they affect the redox state and generate stress-inducing chemical species. This induction of oxidative stress is expected because lutein holds a protection role conveyed by its antioxidant features—particularly under heterotrophic growth, where spontaneous oxidative stress is normally absent (unlike happens with phototrophic cultures) [45].

Finally, the specific growth rate affects both continuous and semicontinuous cultures: lutein tends to accumulate at low dilution rates, but not to levels sufficient to balance the decrease in biomass productivity under such circumstances. Therefore, the maximum lutein productivity is again typically attained at the optimal dilution rate for biomass production [45].

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 L⁻¹ [39]—even though maximum astaxanthin productivities of 11.5 mg L⁻¹ d⁻¹ can be attained at 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 L⁻¹d⁻¹, 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/w_DW) 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 m⁻², with associated productivities increasing from 9 to 13 g m⁻² d⁻¹ 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 m⁻² s⁻¹) 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 μmol_photon m⁻² s⁻¹, were the best conditions for accumulation of astaxanthin—which attained ca. 30 mg g⁻¹.

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 L⁻¹ Fe²⁺-EDTA stimulated synthesis of astaxanthin more effectively, up to contents of 30.7 mg g⁻¹; and despite the lower cell density attained (2.3 × 10⁵ cell mL⁻¹), a higher concentration (36 μmol L⁻¹) 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 L⁻¹ astaxanthin, which correspond to a 28%-increase.

Semicontinuous cultivation of D. salina at 25 °C produced 80 g m⁻³ d⁻¹ biomass [42]—from which 1.25 mg L⁻¹ of β-carotene was recovered [71]; however, this figure could be improved up to 2.45 mg m⁻³ d⁻¹ in continuous biphasic bioreactors [72]. When cultivated photoheterotrophically, a significant increase of cellular β-carotene content was experimentally observed: the maximum score was 70 pg cell⁻¹, in a culture enriched with 67.5 mM acetate and 450 μM FeSO₄ [33].

As with astaxanthin, Fe²⁺ plays an important role in β-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 m⁻² UV-A radiation to 250 W m⁻² PAR stimulated long-term growth of that microalga, and led to a 2-fold enhancement in β-carotene accumulation by 24 d [38].

A major practical problem in using such microalgae as Murielopsis sp. or S. almeriensis is the need for cell wall disruption. This can be accomplished through a variety of ways, e.g., milling, ultrasound, microwave, freezing, thawing or chemical attack [45].

The mortar-and-pestle procedure described by Mínguez-Mosquera et al. [73] provides full recovery, but it cannot be scaled up to industrial practice; sonication and ball milling produce results similar to that procedure, as long as alumina is employed as disaggregating agent [45]. Ceron et al. [74] complemented the alumina-based cell disruption with alkaline treatment using 4% (w/v) aqueous KOH (40 °C); disaggregation and lipid expression were both facilitated.

Microalgal biomass is usually processed via solvent extraction, to render carotenoid extracts—with typical contents of 25% [45]; this can be used directly in the formulation of supplements, or undergo further multistep purification—encompassing hydrolysis to release hydroxylated carotenoids from the accompanying fatty acids, and final recrystallization to polish the product.

Obtaining a carotenoid-rich oleoresin from microalgae—dried or in wet paste form, is a more straightforward task; such extracts may then be subjected to classical processes to obtain purer lutein [45,74] that may successfully compete with that extracted from marigold.