**5. Culturing of Cryptophyta**

Due to their biochemistry, Cryptophytes have applications in aquaculture and a wide variety of biotechnology applications. The most studied species are Pyrenomonadales of the Pyrenomonadaceae family, Rhodomonas/Storeatula/ Rhinomonas, and from the family of Geminigeraceae, Geminigera, Teleaulax, Guillardia, Proteomonas.

## **5.1 For aquaculture**

At the experimental level, cryptophytes have been cultivated using batch systems to determine the effect on the growth of environmental parameters, temperature (12–32°C) [91–94], light in quality (white, blue, green, and red) [94, 95] and quantity (11–600 μM m−2 s−1) [91, 93, 94, 96–98], nitrogen sources (nitrates, ammonium, urea) [90, 99, 100] and quantities [92, 96], all of this has been carried out to optimize the culture under laboratory conditions (**Figure 3**).

**Figure 3.** *Photographs of Proteomonas sulcata cultures in a batch system.*

Cells are easily cultured in small volumes and can reach cell densities of 4–6 × 106 cells ml−1; they are considered an important food source for use in aquaculture since the biomass has a high content of proteins, lipids, fatty acids (PUFA, HUFA), and sterols; with an EPA/DHA ratio close to two [22, 101, 102]. The high nutritional value was especially recognized for copepods [98, 102–105] and mollusks [105, 106]. The cryptophytes *Rhodomonas* sp., *R. salina*, *R. baltica*, and *R. reticulata* have been among the most widely used in aquaculture, and their size (5–20 μm) allows them to be ingested by copepods (medium and adult sizes) that prefer these to other algal groups (Diatoms or Chlorophytes) (personal observation), and mollusks in seed stage, juveniles, and adults. Cryptophytes do not have a cell wall, therefore allows for easier digestion and absorption. The most employed medium is f/2- Si [22, 90, 92, 93, 96, 99, 100, 107], followed by B1 [98, 108], L1 [91], Z8 [109], and 2f [94], with average specific growth rates (μ) between 0.48 and 0.88, indicted that growth is slow for these organisms. Optimal temperatures of 19–24°C for cultivation make using these cells less feasible in temperate zones.

For aquaculture, *Rhodomonas* has been cultured in carboys (20 L) as a batch system; it has been reported that bigger scale cultures have presented difficulties, with unexpected plateau phases or even death compared to *Cryptomonas* sp., more stable and predictable [102]. Semicontinuous cultures of *Rhodomonas* sp. (20 L) have been used for aquaculture, with exchange rates of 0.33 every third day [102]. The main limitations for scaling cultures in nutrient sufficiency are the maintaining light conditions and culture in suspension without damaging cells. Continuous cultures of *Rhodomonas salina* and *Rhodomonas* sp. in column photobioreactors (94 L) have been carried out for feeding copepods [108], with exchange rates of 0.46 d−1 and mean cell densities of 2.40 × 106 cells ml−1. The main changes in those systems were: a smaller column diameter (0.2 m), agitation by supplying air mixed with CO2 from the bottom, and in wider columns (0.8 m and 500 L), the central and external illumination of indoor cultures with different strains (*Teleaulax amphioxeia*—TA, *Rhinomonas* sp., *Chroomonas* sp.) [90]. In this last, cell densities were lower than previously reported (4.6–6.4 × 105 cells ml−1). However, the percentages of EPA and DHA (of TFA) were very stable during the stationary phase, higher for TA (14.6–11%) with slight variations depending on the culture medium (f/2 or urea compound fertilizer), which confirms its high nutritional potential.

The first to work on continuous cultures of *Rhodomonas* sp. using a photobioreactor tubular (200 L) in a greenhouse with natural light [109] showed biomass concentrations up to 1.5 g L−1, 2–5 times higher than previous reports [108]. These results suggest that outdoors cultures can be a good strategy for improving the productivity of cryptophytes and could be a field to open these cells to more biotechnological applications.
