Cryptophyte: Biology, Culture, and Biotechnological Applications

Abstract

Cryptophytes are single-cell biflagellate algae, with extrusive organelles called ejectosomes. They live in fresh and marine water, mainly in shaded environments where light levels are reduced relative to the surface. They are the product of a secondary endosymbiosis of a red alga, which still retains the endosymbiont nucleus’s reminiscences and has four membranes around its plastids. Cryptophytes have a metabolic diversity that makes them very interesting from a nutritional point of view since they present a balance of fatty acids w3/w6, sterols, carotenoid pigments, and phycobiliproteins, these last also have antioxidant effects. Their composition makes them attractive for food in aquaculture and human consumption, pharmaceuticals and cosmetics; their fluorescent potential has attracted the attention of researchers in genomics, neuroscience and molecular biology. The biochemical composition of the cells is modulated by illumination, available nutrients, and its growth phase. This work reviews the general biology of cryptophytes, emphasizing the photosynthetic ones, culture properties and its biotechnological potential.

1. Introduction

Cryptophytes or cryptomonads are eukaryote algae that are biflagellate and unicellular, with sizes between 3 and 50 μm, most are photosynthetic and motile, and a few are palmelloid and form colonies surrounded by mucilaginous sheaths [1]. They are classified into the kingdom Chromista, phylum Cryptophyta, class Cryptophyceae, and order Cryptomonadales. They live in environments from fresh to marine water usually of good quality [2, 3]; also they can be found at varying light conditions and at different temperatures, including those that are extreme such as the Antarctic [4], blooms of cryptophytes have been reported in fresh [5] and marine waters [6]. Cryptophyte’s genus Goniomonad lacks plastids; they are heterotrophic and feed on bacteria and small organic particles. Due to their small size and biochemical composition, Cryptophytes are essential contributors to the food chains of a diversity of organisms [7, 8, 9, 10]. Some cryptophytes are plastid donors for dinoflagellates like Dinophysis acuminata [11] and ciliates like Mesodinium rubrum and M. major [12]. Their importance has been underestimated mainly due to their delicate structure, which can be easily altered or broken by common fixatives such as Lugol and formalin. However, cryptophyte inter-species morphology is not that different allowing for species-level taxonomy by light microscopy [13, 14].

The abundance of cryptophytes is increasing in places like the Antarctic Peninsula [6] and Chesapeake Bay [12], where a notable change has been observed in species composition and size distribution, significantly influencing local ecosystems.

There is general agreement that cryptophytes evolved from a secondary endosymbiosis, which occurred by the engulfment of a red alga by an unknown eukaryote [15, 16]; this event resulted in a cell with two nuclei, two cytoplasms, one of each is in the chloroplast, which is covered with four membranes, and with unique content and distribution among algae of harvesting-light pigments, they have chlorophylls a and c₂, phycocyanin (PCY) or phycoerythrin (PER) [17, 18]. The cryptophytes are complex cells with specific movements and unique structures that allow easy recognition. The main morphological characteristics are slightly ovoid asymmetric cells, two asymmetric flagella with mastigonemes bipartite, an internal and an external periplast plate that surrounds the cell membrane, the structure of the furrow/gullet (groove/throat) [19], ejecti- or ejectosomes that allows them to suddenly alter its swimming direction in the opposite direction [1, 3, 8, 20].

The composition of the cryptophytes, especially in fatty acid, phycobiliproteins (PBPs), and carbohydrates, has attracted the attention of aquaculture and diverse business sectors, such as pharmaceutical, nutraceutical, chemical, and cosmetic industries [21, 22]. The culture of these cells and the production of these substances have some difficulties to overcome, as these delicate cells have a low growth rate compared to other cells in the market.

This chapter will address the Cryptophytes, mainly focusing on those that are photosynthetic, observing their biology, biochemical composition, culture systems, and some of their products with antioxidant potential, as well as fluorescent pigments which are of increasing interest as a marker in biotechnology applications.

2. Morphology, biology, and function

The cell morphology is flatter on the ventral side and concaves on the dorsal side (Figure 1a); the cell shape is like a bean or a drop of water; the form is mainly influenced by the furrow/gullet complex located at the anterior part. A gullet, or a furrow, or some combination of both (Figure 1b) is one of the main diagnostic features of the genera [23]. The complex can be localized by light microscopy in big cells, by the presence of big ejectisomes surrounding it; in small cells, an electron microscope is necessary [9]. The furrow is a ventral groove of variable length that begins in the vestibular region and extends posteriorly to half of the cell. The gullet is an invagination that extends to the posterior side; it originates from surrounding the furrow when it is present, but when the furrow is absent it is found on the ventral side near the vestibulum [8, 20, 23]. The vestibulum is a structure at the anterior side, present in all cells, an outwardly facing depression, from which two asymmetric flagella have a subapical origin on the ventral right side; it is connected to the beginning of the furrow/gullet complex.

Figure 1.

General drawing of a cryptomonad cell. A. Ventral view. B. Side view showing part of the inner periplast component. C—chloroplastid, CER—chloroplastid endoplasmic reticulum, CM—cell membrane, CV—contractile vacuole, DF—dorsal flagellum, F—furrow, G—gullet, GA—Golgi apparatus, IPC—inner periplast component, LE—large ejectisome, LV—lipid vesicle, Mg—mastigonemes, Mi—mitochondrion, Nm—nucleomorph, No—nucleolus, Nu—nucleus, PS—periplastid space, Py—pyrenoid, Ri—ribosome, SE—small ejectisomes, SG—starch granule, TH—tubular hair, Thy—thylakoid, TTF—thin terminal filament, V—vestibulum, VF—ventral flagellum.

2.1 Evasion and defense

Ejectisomes are present in all cryptophytes and are located on the furrow/gullet complex underlying the periplast around the cell, the size of these organelles can be big (500–700 nm) or small (250–250 nm) [24]. Ejectisomes are cylinders-like structures formed by two tightly spiraled taper tapes of unequal size, coiled together and surrounded by a membrane. When the organisms are stressed by sudden changes in pH, osmolarity, or light intensity, ejectisomes are discharged, and the cell can be pushed backward, showing jerky movements [8, 9, 19, 25], probably by the impact of the ejectisomes with an object, it is a defense mechanism that allows the cell to escape from predators [26]. The ejected ejectisomes are tapes unrolled that look like long ribbons with ruffled edges; the big ones can be 7 μm × 228 nm and small ones 3.6 μm × 50 nm [24]. Ejectisomes are synthesized in the Golgi apparatus and are degraded in cytolysosomes during nutrient starvation [27].

2.2 Protection and support

Cryptophytes do not have a cell wall, but the cell membrane is covered and sandwiched by both an inner and surface periplast layer (Figure 1A and B), observed only by electron microscopy. The inner periplast component (IPC) or epiplast can be flat or consist of several plates of different forms, among the shapes identified are polygonal, rectangular, and hexagonal [9, 14, 28, 29]. The surface periplast layer is formed by plates and rosulate scales; however, in some a fibrous coat can be present instead. The epiplast is made of epiplastin, a proteinaceous substance that provides flexibility and protection to the cell membrane [30]. The periplast plates decrease in size toward the rear of the cell and disappear at the furrow/gullet complex and vestibulum [9, 31].

2.3 Motility

The motility of these cells is due to two asymmetric flagella and its cellular form. Both flagellums have rows of mastigonemes similar to those of stramenopiles [32]. The longest flagellum directed to the front has two rows of bipartite hairs, and the shortest flagellum directed to the back only has one, they can be observed by electron microscopy, and structure variations have been described [28]. The bipartite hairs are composed of a tubular attached to the axoneme, it terminates with a single non-tubular filament in the longest flagellum, but in the case of the shortest, it ends with two unequal terminal filaments [9, 20, 28]. In addition, delicate seven-sided scales measuring 140–170 nm in diameter are commonly attached to the hairs [33]. Other structures that are part of the mechanism for motility are the rhizostyle and a compound rootlet system. The rhizostyle is a peculiar microtubular flagellar root that originates near one basal body and extends toward the posterior extreme of the cell; in some species until the nucleus, without a physical connection, but in others, just to the first third of the cell and has a wing-shaped lamellar projection [29, 34]. The cryptophytes have a phototaxis response that is mediated by a two-rhodopsin-based photosensory mechanism, similar to what is observed in green flagellated algae Chlamydomonas reinhardtii [35]. The structure of this mechanism includes an integral membrane protein, with a seven transmembrane alpha-helices covalently bonded to the retinal chromophore to make a channel structure, it is the anion channelrhodopsin, which is light-gated, and initially discovered in chlorophyte algae, which serve as photoreceptors to guide phototactic orientation [36, 37], this structure has been utilized in optogenetic applications [38].

2.4 Control of volume and osmolarity

The Cryptophytes do not have a cell wall, so variations in osmolarity could induce turgidity causing it to burst or plasmolysis causing the cell to compress. However, they possess a remarkable organelle, a contractile vacuole (CV), which has a rhythmic activity with diastolic and systolic cycles allowing for filling and emptying of CV, respectively [39]. This rhythmic mechanism maintains the cell volume and osmolarity, the cycle last either10s in freshwater or 40s in marine water [40]. The CV is near where the flagella structure originates and functions usually by discharging excess water and ions to the vestibulum [41]. Cryptophytes and red algae also employ another mechanism for osmolarity and volume control, synthesis of floridoside (2-O-D-glycerol-α-D-galactoside); which is a low molecular weight carbohydrate that functions as an osmolyte [41, 42, 43]; a similar mechanism is used by red algae algae’s what signals an endosymbiotic inheritance [41].

2.5 Plastid of the cryptophytes

The plastids in alga and plants evolved from the endosymbiosis of a cyanobacterium, which means the incorporation of one cyanobacterium in a heterotrophic cell [44]; this primary endosymbiosis explains the plastid origin in chlorophytes, glaucophytes, and red algae. Other algae stramenopiles, haptophytes, and cryptophytes are the product of a secondary endosymbiosis by an unknown eukaryotic host and a red algal symbiont (Figure 2). Many scientific works confirm this hypothesis [45, 46]. These organisms possess four membranes around the plastid; the outermost membrane is thought to be the phagocytotic vacuole membrane that endocytosed the red algae and evolved to become the chloroplastic endoplasmic reticulum (CER) [47]. The CER is contiguous to the exterior nuclear membrane [9, 19, 48] (Figure 1), it involves the two outer membranes of the plastid, and has ribosomes on its outer surface (Figure 1) [49]. However, in contrast to other algae, the plastid of cryptophytes is more complex [48, 49]; between its two outer and two inner membranes, there is a space that is thought to correspond to the remains of the endocytosed red algae cytoplasm, it is called periplastid compartment (PC). One of the significant adaptations for endocytosis was the loss of genetic information of the endocytosed cell (from the nucleus and chloroplast of the red algae). However, not all nuclear information disappeared in Cryptophytes and Chlorarachniophytes, as occurred in all other secondary endosymbioses, which were sent to the host nucleus by endosymbiotic gene transfer [50, 51]. The remanent of the nuclear information is harbored in the PC and constitutes consists of a small nucleus or nucleomorph (NM). The PC also harbors eukaryotic ribosomes, and numerous starch globules produced over the pyrenoid are visible in the PC (Figure 1A) [49]. The plastids of the cryptophytes require that most of their proteins be nucleus-encoded, and are synthesized as precursors in the cytosol, and subsequently imported through the four membranes surrounding the plastid [47, 52]. This is possible because there is a mechanism for importing proteins that allows crossing of 2–5 membranes when the information is sent to the PC, stroma, or the thylakoid lumen. There should also be a mechanism for the retrograde pathway, from plastid to other organelles [47]; the CER functions are associated with this pathway [53, 54]. Other CER functions are related to bidirectional lipid and metabolite transfer and division [55]. The proteins directed to the plastid are synthesized as preproteins with a bipartite N-terminal signal sequence, which is used for a co-translational translocation of them across the outermost membrane, and after passing this membrane, the signal sequence is cleaved off [53]. The mechanism for passing the second membrane is possibly like the other four membrane plastids. The cryptophytes possess a nuclear-encoded symbiont-specific ERAD machinery (endoplasmic-reticulum-associated protein degradation) and also SELMA (symbiont-derived ERAD-like machinery); the origin of these mechanisms is unclear but is being studied [47]. To reach the stroma, Toc-Tic machinery (translocon of the outer and inner membrane of chloroplasts) similar to that of chlorophytes and diatoms may need to be present; this machinery existed in the common ancestor of all Archaeplastida, organisms with primary plastids [47, 51, 53].

Figure 2.

Secondary endosymbiosis of a red algae that evolved in a cryptomonad cell. The cryptomonads have four membranes plastids; the outer is the plastid endoplasmic reticulum which surrounds plastid and nucleus. The phycobilisomes disappeared, and only one pigment not organized in a structure that is in the thylakoid lumen. Nu—nucleus, No—nucleolus, Thy—thylakoid, Nm—nucleomorph, C—chloroplastid, Ri—ribosome, CER—chloroplastid endoplasmic reticulum.

All cryptophytes have one NM in each PC (Figure 1) with a double membrane with pores similar to those in the nucleus and three chromosomes that NM replicates in coordination with the nucleus [9, 44, 56]. The understanding of the presence of NM could resolve some fundamental questions such as the phylogeny of other algae with secondary plastids that also lack this vestigial structure [48]. The position of the NM is characteristic of the Cryptophyte species [9, 49].

2.6 Pigments and light harvesting by red algae endosymbiont and cryptophytes

Like all phytoplankton, cryptophytes have chlorophyll a as the primary light-harvesting pigment [57], and other accessory pigments ∝-carotene, alloxanthin, chlorophyll c2, and the PBPs for the capture of low light intensity in wavelengths not well absorbed by chlorophyll a (500–650 nm) [17, 58, 59].

The red algae and cyanobacteria have a color that depends on the predominance of PBPs, the orange-red PER or the blue PCY, which are in several hundred and are highly organized in supramolecular complexes, the phycobilisomes (PBS) (Figure 2). The PBS are their main light-harvesting antennae and cover the stromal surface of thylakoids [60]; the PBSs have mobility that lets them distribute the absorbed energy between photosystems (PSI and PSII) [61]. PBPs are composed of two kinds of α and β protein subunits and are more stable in trimer (3α + 3β) or hexamer (6α + 6β). The protein part of apoprotein is covalently bonded to a chromophore or phycobilin [32, 60, 62], these chomoproteins are united to colorless linker polypeptides [63], and constitute the light-harvesting antenna for transferring energy to chlorophyll a to PSII and possibly to PSI [32, 60, 62].

Like red algae, cryptophytes have PBPs pigments, but they do not have PBSs, and, they only produce one kind of PBP pigment per cell (Cr-PE or Cr-PC), packed into the thylakoid lumen [17, 64] without any arrangement [32, 65, 66], it gives the cell a red or blue color [9, 18, 19], but cells possess other accessory pigments, allowing them to display a great diversity of colors [67]. The ratio of Chlorophyll a: PBPs of cryptophytes can be several times higher than that of non-PBPs pigments [18]. The endosymbiosis provided the cryptophytes with new machinery that allowed diversification of light capture [65]; the PBPs are an auxiliary or second light-harvesting system, allowing them to occupy light spectra niches for more efficient light capture [65].

The PBPs of the cryptophytes are composed of two α and two β protein subunits and four linear tetrapyrrole chromophores or phycobilins covalently bonded by one or two thioether bonds to specific cysteine residues on the protein [68]; these PBPs provide unique spectral properties of absorption and emission fluorescence (Table 1).

PBPs	Color	Absorption (nm)	Emission (nm)	Cryptomonad genera	References
Cr-PE 545	Red	538–551	580–587	Teleaulax, Plagioselmis, Geminigera, Hanusia Guillardia, Rhinomonas, Pyrenomonas/Rhodomonas, Storeatula, Proteomonas, Cryptomonas, Baffinella	[13, 18, 67, 69, 70, 71]
Cr-PE 555	Red	553–556	578	Hemiselmis	[69]
Cr-PE 566	Red	563–567	600–619	Cryptomonas, Baffinella, Chilomonas Campylomonas, Falcomonas	[13, 18, 65, 70]
Cr-PC 564	Blue	557–566	N. R.	Hemiselmis	[72]
Cr-PC 569	Blue	568–569	650, 656	Falcomonas, Hemiselmis	[13, 69, 73]
Cr-PC 577	Blue	576–578	634–641	Hemiselmis	[18, 74]
Cr-PC 615	Blue	612–615	580–589	Hemiselmis subgen. Plagiomonas	[18, 69, 70]
Cr-PC 630	Blue	625–630	648, 649	Chroomonas	[17]
Cr-PC 645	Blue	641–650	654–662	Chroomonas, Komma	[69, 70, 73]

Table 1.

Different classes of phycobiliprotein (PBPs) in cryptophytes, spectral ranges of the main absorption and fluorescence emission maxima at visible wavelengths (nm) (Cr, cryptophytes; PE, phycoerythrin; PC, phycocyanin); and the actual Cryptomonad genera.

Adapted from table VI [67, 68]. The PBPs are named, including the wavelengths of maximum absorption [9, 17].N.R. = no reported.

The number and location of phycobilins within the protein are the primary factors that determine the visible absorption, the fluorescence spectrum, and the energy transfer pathway for any given PBP [66]. The complex of chromophores and protein subunits is a complete light-capturing unit; the α subunits of PBPs are encoded in the nuclear genome (derived from the ancestral host), whereas the β subunits are encoded in the plastid as in red algae, so the PBPs are unique chromoprotein complexes that originated from secondary endosymbiosis [17, 65]. Another function of PBPs in cryptophytes is to help them with photoacclimation; this process involves changes in PBPs concentrations and shifts in the PBPs absorbance peaks when they are grown under red, blue, or green light [58]. The anterior means the photosynthetic system of cryptophytes is very different from other algae [75, 76], the location of PBPs in thylakoid lumen, the presence of dimeric PBPs forms, and the principal connection to PSII, but the precise mechanism remains to be discovered.

Another structure in the chloroplast is the pyrenoid, where the enzyme RUBISCO responsibly for CO2 fixing is located, there is one pyrenoid per plastid, and its position is an identifying characteristic of each species (Figure 1) [31].

3. Isolation, maintenance, and availability of cryptophytes

Cryptophytes are inhabitants of still waters with a low trophic state, they reach maximal biomass near the summer chemocline [77], and vertically migrate as a mechanism for harvesting inorganic nutrients; they can grow in turbid waters with low light due to their efficient greenlight harvesting PBPs [64]. Nets of different kinds and sampling bottles can be used for collecting cryptomonads from the water column; sediments or mucilage should be placed in the water, so the cells can be motile while transporting them to the laboratory, and all samples should be placed at a low temperature [20, 77]. In the lab, the samples should be gently filtered (>30 μm–<200 μm) and cultured by enriching the water with one standard culture media diluted (1:5–1:10), WARIS, and BBM (freshwater); ASP-12, ESM, f/2, and ASP (marine; check recipes on the CCAC, CCAP, CCMP, NIES, and SAG websites) [9, 78]. Cryptophytes do not grow in agar (only the palmella-forming taxa). Fluorescent light (20–50 μmol/m²/s), 16:8 or 12:12 light:dark cycle, and 16–25$°\mathbf{C}\text{.}$It is recommended that culture conditions should be the same or as close as possible to that of the sampling site. When cells establish a population, they can be isolated, and the method of choice is using a micropipette [9, 79]; another way is by dilution in a sterile 24-well microplate. The delicate cells do not resist the fixation with Lugol or formol, but glutaraldehyde 2% can be an option [9, 80, 81]. The cells can be identified as cryptophytes using a standard optical microscope, with respect to size, form, and movement. Photographs are difficult to obtain because of cell movement, but dark field, phase contrast, differential interference contrast (DIC), and fluorescence microscopy help identify some features of cells [9, 20]. For maintaining the isolated cells, low temperature and low light are recommended. With heterotrophic cryptophytes like Goniomonas and Chilomonas, an organic carbon source, like a sterilized wheat seed, pea, or lentil, in a soil-water media are typically provided. Some also grow in WARIS added with soil extract [9, 20]. Different strains of cryptomonads are available in collections around the world.

4. Taxonomy

Although distinguishing cryptophyte cells from other flagellated cells is relatively easy by their movement, gross morphology, and epifluorescence; classifying by genus and species is difficult [9, 82]. Initial investigations proposed the morphology and color as a basis for this definition [20, 23, 25], but color changes depending on the cell stage and culture conditions, mainly light and nitrogen [83, 84]. Characteristics such as the groove/gullet complex, or ultrastructural features such as flagella, the position of NM, pyrenoid, and IPC, can only observed with an electron microscope, but could be characters of taxonomic value. However, this is view has changed after discovery that some species with haploid and diploid phases can have very different morphologies, suggesting that there is an intermediary stage sexual reproduction that could be confounding taxonomic certainty of species [9, 31, 81].

Among the characteristics used in Cryptophyte systematics are the presence or absence and the kind of accessory pigment (PER or PCY) (Table 1) [23, 28, 33]. The ultrastructural traits such as the arrangement of flagellar hairs [28], the number and location of the NM in the PC [1, 23], the presence and location of the eyespot in Chroomonas species, the type of scales comprising the outer periplast component, number location, and type of pyrenoids [8, 23], number of chloroplast per cell [85] are some of the taxonomic keys still used. Advances in molecular tools improved the phylogeny of the groups. There is a correspondence between molecular data with biliprotein, but it has not found with other morphological traits like IPC.

Life histories may be another character. Although initially it was thought that all cryptophytes divided only vegetatively, now species such as Proteomonas sulcata have been identified to have a dimorphic life cycle [86], Cryptomonas/Campylomonas [83], Teleaulax/Plagioselmis [13], are known to have complex life cycles. In some cases, those species have shown alternation of generations, with very different forms [10] and haploid/diploid phases. They had even been classified as different species. The aforementioned shows how complicated the taxonomic classification of cryptophytes can be.

Few of the genes more employed in the cryptophytes phylogeny are nuclear, 18S, ITS1, 5.8S, ITS2, 28S, SSU, LSU rDNA [13, 70, 87]; the nucleomorph SSU rDNA and 18S rDNA [83], and the chloroplast psbA [32].

The Cryptomonad classification of the Phylum Cryptophyta is shown in Table 2; however, this table will change in the future to reflect advances in knowledge, culturing, and electron microscopy.

	References
Class Goniomonadophyceae
Order: Goniomonadida
Family: Goniomonadaceae; Genus: Goniomonas
Class Cryptophyceae
Order: Cryptophyceae
Family: Cryptomonadaceae; Genus: Cryptomonas (includes Campylomonas/Chilomonas)	[82, 88]
Order: Pyrenomonadales
Family: Pyrenomonadaceae
Genus: Rhodomonas/Storeatula/Rhinomonas	[89]
Family: Geminigeraceae
Genus: Geminigera/Teleaulax/Plagioselmis, Hanusia, Guillardia, Proteomonas	[13]
Family: Chroomonadaceae
Genus: Chroomonas, Falcomonas, Komma	[88]
Family: Hemiselmidaceae
Genus: Hemiselmis

Table 2.

Cryptomonad classification of the phylum Cryptophyta Cavalier-Smith emend.

Clay, Kugrens and Lee [23, 90]. The color of the genus indicates the kind of pigment it contains. Red = Cr-phycoerythrin, Blue = Cr-phycocyanin, Black = No color = no photosynthetic, no chloroplasts present.

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, 92, 93, 94], light in quality (white, blue, green, and red) [94, 95] and quantity (11–600 μM m⁻² s⁻¹) [91, 93, 94, 96, 97, 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, 103, 104, 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.

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, 111, 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]:

$$C=\frac{A}{\epsilon \ast d}\times MW\times \frac{Vb}{Vs}\times \frac{{10}^{12}}{N}$$E1

Where A = absorbance of sample, $\epsilon$ = 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) Vb and Vs = 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.

6. Biotechnological applications

Cryptophytes are cells with unique biochemistry; for example, it has a high and balanced content of PUFA (ALA, SDA, EPA, DHA) [21, 22], phytosterols, carbohydrates [21], and fluorescent pigments PBPs. Each of these compounds has effects on health [21, 22, 83, 120], giving them a wide variety of potential applications in food, cosmetics, pharmaceuticals, medicine, immunology, as well as for scientific research [116]. There are many patents for these applications, but most are related to fluorescent properties of PBPs [121].

The food and beverage industries have increased attention to natural pigments from microorganisms, especially aquatic products from macro and microalgae [21]. This trend is predicted to continue in the future due to increasing consumer health consciousness of potential and known harmful effects of synthetic compounds. Other advantages are stability, considering temperature, pH, osmolarity, low reactivity [122, 123, 124], and the health-promoting effects from another point of view [121]. An advantage is that the market for cyanobacteria and their PBPs (mainly Arthrospira,Anabaena) and red alga (Porphyridium and Galdieria) has been established. For PBPs and their products, in 2010, it was estimated to be around US$60 million [125]. Another critical factor for industries is the stability of PBPs, they showed good range of stability between pH 5–10 and between −20 and 80°C, and it could be prolonged by adding preservatives [124]. The blue (PCY) and red (PER) pigments that Cryptophytes provide is attractive to the food and cosmetic industries [21]. Some of their uses are in chewing gum, desserts, candies, dairy products, ice cream, soft drinks in different presentations (aqueous and alcoholic), and cosmetics like lipstick and eyeliners [116, 119, 126, 127]. Some applications like food and cosmetics do not need high purity, thus semi-purified pigments are an attractive option since they are less expensive than highly purified pigments, that are needed for using them as molecular markers. The purity is measured by the ratio of the maximum pigment absorbance divided by its absorbance at 280 nm; for food, a ratio of ≥0.7 is accepted, a reagent grade is ~3.9, and analytical grade ≥4.0 [117], that is the reason why PER is expensive and sold anywhere from US$200 to US$100,000/kg.

In regards to the effects of PBPs, some are related to their antioxidant potential [119, 128, 129] as they are natural ROS scavengers, have anti-inflammatory [130, 131], and anti-aging properties [119, 128, 129]. Other protective effects of PBPs are located in mitochondria membrane from ROS stress, which could maintain cellular viability and proliferation [131, 132] in different health problems. PCY also has a pro-apoptotic effect in different cancer cell lines and is an inhibitor of COX-2 enzyme, which converts arachidonic acid to prostaglandins and plays a crucial role in tumor progression and chemical resistance, and the PGE2, which participates in promoting angiogenesis [132]. PCY has been observed as an inhibitor of viral protease activities (i. e. SARS-CoV-2) [133, 134], which could mean antiviral protection. The anti-diabetes activity of phycocyanobilin could be due to the inhibition of NADPH oxidase and the protective effects against human lymphatic endothelial cells apoptosis, which also explains its neuroprotective effects [135].

PBPs play an essential role in fluorescent-based detection systems, like with flow cytometry, epifluorescence, and confocal microscopy for fluorescent immunoassays like protein electrophoresis [136], immunophenotyping; they can also be used as selective markers of specific biological structures, i.e., arterial wall thickness, atherosclerotic plaque, luminal boundaries and to better delineate tumors mass outlines and such other fluorescent studies [121, 137]. Spectral properties, such as excitation and emission at the red end of the spectrum and diminished interference from biological matrices give it considerable stability for quenching compared to other biological compounds. In addition, PBPs have high water solubility with minimal interaction with other substances, and the ease of binding to antibodies by conventional cross-linking reagents, have made these fluorochromes unique and superior to other products, i.e. for medical tagging [137, 138, 139]. All of this is motive for developing new biotechnological processes and products, and the commercialization of new patents is still a goal in the near future.

7. Conclusion and prospects

The cryptophytes are secondary endosymbionts that inherited many red algae characteristics, but differences involving morphology and physiology make them unique and not completely understood. The functioning of PBPs as a secondary antenna into the thylakoid and its connection to photosynthesis, the PBPs as an energy reserve, to understand why these cells still have a vestigial nucleus NM with three chromosomes, the way the nucleus coordinates the activities of NM and plastid are some of the mysteries for the scientist to solve. From an applications point of view, cryptophytes have high nutritive value that includes a balanced PUFA profile, high protein content, and the possibility to induce a high content of PCY or PER, antioxidant pigments, as well as having a very stable fluorescence making it attractive for use in research, thus its future looks promising and applications using cryptophytes should grow. From a production perspective, these cells have some advantages compared to other producers of PBPs, they have only one type of pigment. Cryptophytes lack of a cell wall facilitating digestion and nutrient absorption by organism and the extraction of products, and in addition have high content of PBPs with a lower molecular weight. The low growth rates and how to achieve the scaling necessary for high biomass with high pigments content will continue to be a challenge in the near future. Improvements will come probably by improved understanding of its physiology. Also, needed is to increase the variety of organisms in collections, improve the culture procedures including photobioreactors, and finally make more accessible purification methods.

Acknowledgments

The author thanks Gerardo Hernández García for the Figures 1 and 2 and Dr. Miguel Victor Cordova Matson for his support with edition. This work was supported by institutional projects CIBNOR-20427 and CIBNOR-20437.