**Table 2.** Optimal conditions of production of carotenoids by selected microalgae.


**Table 2.** *Cont.* 

**AF**: air flow; **LDC**: light/dark cycle; **LI**: light irradiance; **MM**: metabolic mode; **SR**: stirring rate; **T**: temperature. 

More advanced technologies have meanwhile been made available pertaining to closed systems; these provide better options for growth of most microalgal strains, by protecting the culture from contamination by unwanted (and often ill-defined) microorganisms, and allowing comprehensive and integrated control of processing conditions. Such photobioreactors are either flat or tubular, and may adopt a variety of designs and operation modes. They lead to higher volumetric productivities and an overall better quality for the biomass (or product) generated—but they are also more expensive to build and operate than their open counterparts [9]. 

Some microalgae exhibit unique productivity and plasticity features: when grown under distinct sets of operating conditions, they may accumulate different products to high levels; hence, careful design and control of medium composition, temperature, pH, aeration, stirring and irradiance are recommended. A few examples of optimum conditions of operation of microalgal reactors—using productivity of carotenoids as objective function, are listed in Table 2. 

During microalgal cultivation, a few processing parameters can be specifically manipulated for maximum synthesis of carotenoids; the better studied cases are lutein, astaxanthin and Ά-carotene—which will be discussed below at some length**.** 

## *5.1. Lutein*

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., Ά-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]. 
