Most plants and algae filo contain chlorophyll a and chlorophyll b to harvest light energy. As chlorophyll a is active at wavelengths 430 and 680 nm, and chlorophyll b at wavelengths 450 and 660 nm, these plants and algae are photosynthetically active within this range. However, since red seaweeds only have chlorophyll a, light is only harvested within the blue and red region of the visible spectrum, and there would be an absorption gap in the spectra region in between ^[1][17]. In order to fill this gap and optimize light harvests, red seaweeds assemble phycobilissomes (PBS) in the thylakoid membrane. PBS are highly efficient, supramolecular complexes, with an absorption range of 500–660 nm, which capture solar energy and transfer it to photosystems. The role of PBS as the main light-harvesting chromoproteins was discovered in 1883 by Theodor Wilhelm Engelmann ^[2][18], through the study of the Cyanobacteria Oscillatoria. Nowadays, however, it is known that the PBS role extends beyond light-harvesting, it being also acknowledged to hold an important task as photo-protectors against high irradiances, as well as a nutrient source in times of nitrogen and phosphorus insufficiency ^[3][19].

Phycobilissomes can be classified into three types according to their morphology (hemi-ellipsoidal, hemi-discoidal, and bundle shaped) ^[4][1] and are composed by phycobiliproteins (PBP). PBP present an intrinsic brilliant color being highly fluorescent ^[5][20], and can be found not only in Rhodophyta (comprising up to 50% of all water-soluble proteins) ^[6][21] but also in Cyanobacteria (comprising up to 60% of all water-soluble proteins) ^[7][22] and in Cryptomonads (phylum Cryptophyta) ^[8][23]. PBP are classified in different families, according to absorption properties, and present a distinctive color conveyed by the chromophores: the red–pink phycoerythrin (PE, λ max = 540–570 nm), the blue phycocyanin (PC, λ max = 610–625 nm), the blue–green allophycocyanin (APC, λ max = 650–660 nm), and the blue–pink phycoerythrocyanin (PEC, λ max = 560–600 nm, not present in red seaweeds) ^[9][1][10][4,17,24], along with hydrophobic linker peptides ^[4][1]. All these PBP are generally composed of an α and a β subunit, and among these, PE also holds a γ subunit ^[11][25]. These subunits may contain about 160 to 180 amino acid residues, and are connected with prosthetic group chromophores, which are essentially linear tetrapyrrole groups that bind an apoprotein to cysteine residues through a thioether bond ^[9][12][4,26]. The protein to which this attachment occurs determines the PBP absorption spectrum, and the prosthetic group of the chromophore. A phycobilin can be categorized as phycoerythrobilin (pink–red compound), phycocyanobilin (blue compound), phycourobilin (yellow compound), and phycoviolobilin (purple compound) (Figure 1) ^[9][4]. PE and PEC are rich in blue-absorbing phycoerythrobilin, phycourobilin, and phycoviolobilin, whereas PC and APC contain phycocyanobilin only ^[13][27]. All PBP are tasked with (1) capturing incident light, and (2) participating in the energy transfer chain. This energy transfer is unidirectional, highly efficient (greater than 90%) ^[14][28], and allows red seaweeds to harvest a much wider range of light wavelengths than the other groups of seaweeds ^[1][17] and thus, optimize their photosynthetic efficiency.

In fact, PBP are essential in guaranteeing the growth and adaptation of red algae, by optimizing their light-harvesting abilities in the deeper layers of the water column, where only the blue–green spectrum of the incident light prevails ^[9][4]. Generally, red algae growing under low light have the highest amount of PBP per cell and the highest number of PBS per square micrometer, over red algae growing under high light ^[15][16][6,29], but other factors such as nutrient cycles and diurnal/annual photoperiod can also play a role in shaping the relative pigment content ^[16][17][29,30].

1.2. Biotechnological Potential and Applications

PBPs have noteworthy spectroscopic properties, such as high absorption coefficient, high excitation and emission spectra, high quantum yield, low interference, high quenching stability, and water solubility ^[9][18][4,31]. Therefore, they have been widely considered in several and well documented applications, namely, in biomedical research, clinical diagnostics, therapeutic science, and cosmeceutical and pharmaceutical industries ^{[4][14][19][20]}[1,28,32,33].

However, the primary commercial interest in PBP stems from the fact that these proteins offer health benefits as antioxidants and free-radical scavengers ^{[21][22][23][24]}[5,37,38,39], having a therapeutic and nutraceutical effect ^{[25][26][27][28]}[40,41,42,43], as well as being effective neuroprotective, anti-bacterial ^[22][37], anti-viral ^[29][44], anti-inflammatory ^[30][45], anti-allergic ^[31][46], anti-tumoral ^{[22][32][33][34][35]}[37,47,48,49,50], anti-ageing ^[31][46], anti-Alzheimer, hepatoprotective ^[24][39], immunomodulatory ^[36][51], and hypocholesterolemia agents ^[9][3][27][4,19,42]. In addition to their antioxidant power, PBPs are non-toxic and non-carcinogenic natural dyes. Therefore, they are also earning crescent interest over synthetic colorants within the food and cosmetic industry ^[37][38][6][9,10,21], following consumer demands in their pursue for a healthier lifestyle.

1.3. Extraction and Purification Methods

PBPs are not easy and straightforward obtained. Traditionally, the methods to obtain PBP extracts present a challenge by themselves since, as mentioned, PBP are located within the phycobilissome inside the chloroplast, and thus, the algae must be pretreated with appropriate solvents, and the cells must be homogenized and disrupted using suitable methods, to release the contents within ^[2][18]; this must be achieved while avoiding any step or method that involves high temperatures, as these pigments are highly thermosensitive.

There are different methods to perform the extraction, whose choice is a critical step to attain a maximum PBP recover, as it has a significant impact on activity and purity of the obtained pigment ^[4][1]. Specific factors include biomass conditioning (fresh versus dry algae), biomass/solvent ratio, cellular disruption method, solvent, extraction time and number of steps taken ^[2][18], storage method prior to extraction ^[39][53], and even factors unconnected to the methodology itself, such as the harvest season ^[2][18], and species where the pigment was extracted from ^[40][54].

Traditionally, the abundance and diversity of the different PBP in an organism has been commonly estimated by means of light absorption assessment at distinct wavelengths, performed upon the PBP extracted from the biological matrix ^[41][42][55,56]. However, authors such as Saluri et al. ^[43][57], who lists several other works that reply on this approach to assess the PBP R-PE (a subtype of PE obtained from Rhodophyta ^[14][28]) content in red seaweed species, defend a more reliable method being needed, so that the most promising algae species regarding R-PE content can be targeted. According to the authors, the traditional method of absorbance reading is both quick and widely used; however, it can produce misleading results whenever impurities remain present in the samples, and it is unreliable overall, regardless of whether it is assessed upon crude extracts or following their purification. An example offered in order to circumvent this is found in Saluri et al. ^[43][57]’s work, which describes an alternative approach of employing the High Performance Liquid Chromatography technique of Size Exclusion Chromatography (HPLC-SEC) method with fluorescence and photo-diode array detectors, not only to separate PBP from interfering compounds, but also to reliably quantify their yields.

1.4. Production and Commercialization

As the production of PBP from natural sources requires a high investment in large-scale cyanobacteria or algae cultures, alternative approaches to obtain this pigment for biotechnological purposes has been investigated. Specifically, the production of recombinant PBP in Escherichia coli, while retaining all the qualities of a pigment obtained from native organisms, has been studied by a number of authors ^[20][44][45][33,60,61].

Nevertheless, a few examples can be found on a commercial level, where there are a few companies profiting from PBP commercialization in the form of natural dyes yet featuring prominently microalgae as the source for their products. For example, the commercially available blue colorant Linablue^® Spirulina Extract is sold as a food product decoration component, and several companies promote their microalgae-based natural dyes to be introduced in cosmetic products, namely, phycocyanin from Arthrospira sp.

1.5. Phycoerythrin

Phycoerythrin (PE), the red pigment, owes part of its name to “erythros”, which means “red” in Greek. PE is a photosynthetic pigment, present in red macroalgae ^[46][35], red microalgae ^[8][23] and cryptomonads ^[47][64]. The original source where they are found determines their further classification into R-PE (PE obtained from Rhodophyta, λ max = 545–565 nm, Figure 23 and Figure 34), C-PE (PE obtained from Cyanobacteria, λ max = 540–570 nm), and B-PE (PE obtained from Bangiales, a specific family of filamentous red macroalgae, λ max = 546–565 nm) ^[9][14][4,28]; besides their origin, these compounds have among them slight variations in their absorption characteristics ^[48][62]. PE stands as the major soluble protein produced by the cell of red algae kept growing under low light but high nutrient load ^[49][65].