**5.2 Pigments production (Cr-PE and Cr-PC)**

There are advantages to the production of PBPs from Cryptophytes. The first is that they are water-soluble compounds; furthermore, compared to cyanobacteria and red algae, each species of Cryptophyte produces only one type of PBP pigment (Cr-PE or Cr-PC) (**Table 1**), and the cells are easily broken to free PBPs by freezing and thawing. An inversely proportional relationship relates the PBPs production to irradiance [107, 110]; it is contrary to the biomass, which is directly related [95]. The Cr-PE production (pg cell−1) is also related to wavelength, and has been observed that red light induces a greater production, followed by green light [110] during the exponential stage of culture when there are sufficient nutrients. The cryptophytes studied were *Rhodomonas* sp., *Proteomonas sulcata*, [92, 98, 110–112]; *Guillardia theta*, *R. salina*, *P. sulcata*, *Storeatula* sp., and *Chroomonas mesostigmatica* [69]. The content of PBPs in Cryptophytes can be up to 20% of dry cell weight, which contrasts with other photosynthetic pigments (chlorophyll and carotenoids), which are up to 10 times lower [69]; these PBPs percentages can be reached under the sufficiency of nutrients and low irradiance (20–40 μM m−2 s−1). It is important to mention, when cells lack nutrients, especially nitrogen, the decrease in pigment content is proportional to the nutrient deficit, this fact has suggested that pigments are a nitrogen reserve [84] like in cyanobacteria. Understanding the role of PBPs in the cryptophytes would help solve the problem of high biomass with low pigment or low biomass with high pigment.

The best method for extracting PBPs from biomass, which is obtained by centrifugation, is to suspend biomass in buffer phosphate (0.1 mol L−1) (pH 6, 7–7.2) in accord to [69, 113]. Then cycles of frozen/thaw, homogenize the suspension and centrifugate (11,000 g) to remove the cellular debris, obtain the supernatant; measure the absorbance by a scan (450–750 nm), determine the maximal absorption employing 1 cm quartz cuvette, and use buffer phosphate as blank. Samples not analyzed immediately can remain at −20°C or − 80°C (until six months). For determining the PBPs content (*C* pg cell−1), Eq. (1) is proposed by [69]:

$$C = \frac{A}{\varepsilon \* d} \times MW \times \frac{Vb}{V\mathfrak{s}} \times \frac{10^{12}}{N} \tag{1}$$

Where *A* = absorbance of sample, ε = extinction coefficient, for Cr-PE (5.67 × 105 L mol−1 cm−1) [114] and for Cr-PC (5.7 × 105 L mol−1 cm−1) [115], *d* = path length, MW = molecular weight (Cr-PE =45,000; Cr-PC = 50,000 Da) V*b* and V*s* = volume of buffer and sample, *N* = number of cells L−1.

The extraction mentioned produces a crude extract, which has to be purified for higher quality and value; depending on the purity, PBPs can reach a price of 130 USD – to 15–33 × 103 USD per gram [116, 117]. In general, the purification procedures consist of removing impurities by precipitation with ammonium sulfate, dialyzation, and separation by gel filtration chromatography [118]; in [119], **Table 1** lists various procedures employed for PBPs purification.
