Traditional and new trend strategies to enhance pigment contents in microalgae.

Microalgae are a source of a wide variety of commodities, including particularly valuable pigments. The typical pigments present in microalgae are the chlorophylls, carotenoids, and phycobiliproteins. However, other types of pigments, of the family of water-soluble polyphenols, usually encountered in terrestrial plants, have been recently reported in microalgae. Among such microalgal polyphenols, many flavonoids have a yellowish hue, and are used as natural textile dyes. Besides being used as natural colorants, for example in the food or cosmetic industry, microalgal pigments also possess many bioactive properties, making them functional as nutraceutical or pharmaceutical agents. Each type of pigment, with its own chemical structure, fulfills particular biological functions. Considering both eukaryotes and prokaryotes, some species within the four most promising microalgae groups (Cyanobacteria, Rhodophyta, Chlorophyta and Heterokontophyta) are distinguished by their high contents of specific added-value pigments. To further enhance microalgae pigment contents during autotrophic cultivation, a review is made of the main related strategies adopted during the last decade, including light adjustments (quantity and quality, and the duration of the photoperiod cycle), and regard to mineral medium characteristics (salinity, nutrients concentrations, presence of inductive chemicals). In contrast to what is usually observed for growth-related pigments, accumulation of non-photosynthetic pigments (polyphenols and secondary carotenoids) requires particularly stressful conditions. Finally, pigment enrichment is also made possible with two new cutting-edge technologies, via the application of metallic nanoparticles or magnetic fields.

Narrative Review of the Current and Future Perspectives of Phycobiliproteins' Applications in the Food Industry: From Natural Colors to Alternative Proteins.

Vivid-colored phycobiliproteins (PBPs) have emerging potential as food colors and alternative proteins in the food industry. However, enhancing their application potential requires increasing stability, cost-effective purification processes, and consumer acceptance. This narrative review aimed to highlight information regarding the critical aspects of PBP research that is needed to improve their food industry potential, such as stability, food fortification, development of new PBP-based food products, and cost-effective production. The main results of the literature review show that polysaccharide and protein-based encapsulations significantly improve PBPs' stability. Additionally, while many studies have investigated the ability of PBPs to enhance the techno-functional properties, like viscosity, emulsifying and stabilizing activity, texture, rheology, etc., of widely used food products, highly concentrated PBP food products are still rare. Therefore, much effort should be invested in improving the stability, yield, and sensory characteristics of the PBP-fortified food due to the resulting unpleasant sensory characteristics. Considering that most studies focus on the C-phycocyanin from Spirulina, future studies should concentrate on less explored PBPs from red macroalgae due to their much higher production potential, a critical factor for positioning PBPs as alternative proteins.

Effect of Arthrospira maxima Phycobiliproteins, Rosiglitazone, and 17ß-Estradiol on Lipogenic and Inflammatory Gene Expression during 3T3-L1 Preadipocyte Cell Differentiation.

The study evaluated the effects of Arthrospira maxima phycobiliproteins (PBPs), rosiglitazone (RSG), and 17ß-estradiol (E) on the differentiation process of 3T3-L1 cells and on their regulation of lipogenic and inflammatory gene expression at different stages of the process. The results showed that phycobiliproteins promoted cell proliferation after 24 h of treatment. Furthermore, for all three treatments, the regulation of the highest number of markers occurred on days 6 and 12 of differentiation, regardless of when the treatment was applied. Phycobiliproteins reduced lipid droplet accumulation on days 3, 6, 10, and 13 of the adipogenic process, while rosiglitazone showed no differences compared to the control. On day 6, both phycobiliproteins and rosiglitazone positively regulated Acc1 mRNA. Meanwhile, all three treatments negatively regulated Pparγ and C/ebpα. Phycobiliproteins and estradiol also negatively regulated Ucp1 and Glut4 mRNAs. Rosiglitazone and estradiol, on the other hand, negatively regulated Ppara and Il-6 mRNAs. By day 12, phycobiliproteins and rosiglitazone upregulated Pparγ mRNA and negatively regulated Tnfα and Il-1ß. Additionally, phycobiliproteins and estradiol positively regulated Il-6 and negatively regulated Ppara, Ucp2, Acc1, and Glut4. Rosiglitazone and estradiol upregulate C/ebpα and Ucp1 mRNAs. The regulation exerted by phycobiliproteins on the mRNA expression of the studied markers was dependent on the phase of cell differentiation. The results of this study highlight that phycobiliproteins have an anti-adipogenic and anti-inflammatory effect by reducing the expression of adipogenic, lipogenic, and inflammatory genes in 3T3-L1 cells at different stages of the differentiation process.

Nutraceutical Features of the Phycobiliprotein C-Phycocyanin: Evidence from Arthrospira platensis (Spirulina).

Arthrospira platensis, commonly known as Spirulina, is a photosynthetic filamentous cyanobacterium (blue-green microalga) that has been utilized as a food source since ancient times. More recently, it has gained significant popularity as a dietary supplement due to its rich content of micro- and macro-nutrients. Of particular interest is a water soluble phycobiliprotein derived from Spirulina known as phycocyanin C (C-PC), which stands out as the most abundant protein in this cyanobacterium. C-PC is a fluorescent protein, with its chromophore represented by the tetrapyrrole molecule phycocyanobilin B (PCB-B). While C-PC is commonly employed in food for its coloring properties, it also serves as the molecular basis for numerous nutraceutical features associated with Spirulina. Indeed, the comprehensive C-PC, and to some extent, the isolated PCB-B, has been linked to various health-promoting effects. These benefits encompass conditions triggered by oxidative stress, inflammation, and other pathological conditions. The present review focuses on the bio-pharmacological properties of these molecules, positioning them as promising agents for potential new applications in the expanding nutraceutical market.

Growth phase-dependent reorganization of cryptophyte photosystem I antennae.

Photosynthetic cryptophytes are eukaryotic algae that utilize membrane-embedded chlorophyll a/c binding proteins (CACs) and lumen-localized phycobiliproteins (PBPs) as their light-harvesting antennae. Cryptophytes go through logarithmic and stationary growth phases, and may adjust their light-harvesting capability according to their particular growth state. How cryptophytes change the type/arrangement of the photosynthetic antenna proteins to regulate their light-harvesting remains unknown. Here we solve four structures of cryptophyte photosystem I (PSI) bound with CACs that show the rearrangement of CACs at different growth phases. We identify a cryptophyte-unique protein, PsaQ, which harbors two chlorophyll molecules. PsaQ specifically binds to the lumenal region of PSI during logarithmic growth phase and may assist the association of PBPs with photosystems and energy transfer from PBPs to photosystems.

Monomeric Far-red and Near-infrared Fluorescent Biliproteins of Ultrahigh Brightness.

Far-red and near-infrared fluorescent proteins have regions of maximum transmission in most tissues and can be widely used as fluorescent biomarkers. We report that fluorescent phycobiliproteins originating from the phycobilisome core subunit ApcF2 can covalently bind biliverdin, named BDFPs. To further improve BDFPs, we conducted a series of studies. Firstly, we mutated K53Q and T144A of BDFPs to increase their effective brightness up to 190 % in vivo. Secondly, by homochromatic tandem fusion of high-brightness BDFPs to achieve monomerization, which increases the effective brightness by up to 180 % in vivo, and can effectively improve the labeling effect. By combining the above two approaches, the brightness of the tandem BDFPs was much improved compared with that of the previously reported fluorescent proteins in a similar spectral range. The tandem BDFPs were expressed stably while maintaining fluorescence in mammalian cells and Caenorhabditis elegans. They were also photostable and resistant to high temperature, low pH, and chemical denaturation. The tandem BDFPs advantages were proved in applications as biomarkers for imaging in super-resolution microscopy.

Dehydration, Rehydration and Thermal Treatment: Effect on Bioactive Compounds of Red Seaweeds Porphyra umbilicalis and Porphyra linearis.

The nutritional and bioactive value of seaweeds is widely recognized, making them a valuable food source. To use seaweeds as food, drying and thermal treatments are required, but these treatments may have a negative impact on valuable bioactive compounds. In this study, the effects of dehydration, rehydration, and thermal treatment on the bioactive compounds (carotenoids, phycobiliproteins, total phenolic content (TPC), total flavonoids content (TFC)), antioxidant (ABTS and DPPH radical scavenging activities) and anti-Alzheimer's (Acetylcholinesterase (AchE) inhibitory activities, and color properties of Porphyra umbilicalis and Porphyra linearis seaweeds were evaluated. The results revealed significant reductions in carotenoids, TPC, TFC, and antioxidant activities after the seaweeds' processing, with differences observed between species. Thermal treatment led to the most pronounced reductions in bioactive compound contents and antioxidant activity. AchE inhibitory activity remained relatively high in all samples, with P. umbilicalis showing higher activity than P. linearis. Changes in color (ΔE) were significant after seaweeds' dehydration, rehydration and thermal treatment, especially in P. umbilicalis. Overall, optimizing processing methods is crucial for preserving the bioactive compounds and biological activities of seaweeds, thus maximizing their potential as sustainable and nutritious food sources or as nutraceutical ingredients.

Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction.

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Variability in spectral absorption within cryptophyte phycobiliprotein types.

Cryptophytes are known to vary widely in coloration among species. These differences in color arise primarily from the presence of phycobiliprotein accessory pigments. There are nine defined cryptophyte phycobiliprotein (Cr-PBP) types, named for their wavelength of maximal absorbance. Because Cr-PBP type has traditionally been regarded as a categorical trait, there is a paucity of information about how spectral absorption characteristics of Cr-PBPs vary among species. We investigated variability in primary and secondary peak absorbance wavelengths and full width at half max (FWHM) values of spectra of Cr-PBPs extracted from 75 cryptophyte strains (55 species) grown under full spectrum irradiance. We show that there may be substantial differences in spectral shapes within Cr-PBP types, with Cr-Phycoerythrin (Cr-PE) 545 showing the greatest variability with two, possibly three, subtypes, while Cr-PE 566 spectra were the least variable, with only ±1 nm of variance around the mean absorbance maximum of 565 nm. We provide additional criteria for classification in cases where the wavelength of maximum absorbance alone is not definitive. Variations in spectral characteristics among strains containing the same presumed Cr-PBP type may indicate differing chromophore composition and/or the presence of more than one Cr-PBP in a single cryptophyte species.

Structure of the antenna complex expressed during far-red light photoacclimation in Synechococcus sp. PCC 7335.

Far-red light photoacclimation, or FaRLiP, is a facultative response exhibited by some cyanobacteria that allows them to absorb and utilize lower energy light (700-800 nm) than the wavelengths typically used for oxygenic photosynthesis (400-700 nm). During this process, three essential components of the photosynthetic apparatus are altered: photosystem I, photosystem II, and the phycobilisome. In all three cases, at least some of the chromophores found in these pigment-protein complexes are replaced by chromophores that have red-shifted absorbance relative to the analogous complexes produced in visible light. Recent structural and spectroscopic studies have elucidated important features of the two photosystems when altered to absorb and utilize far-red light, but much less is understood about the modified phycobiliproteins made during FaRLiP. We used single-particle, cryo-EM to determine the molecular structure of a phycobiliprotein core complex comprising allophycocyanin variants that absorb far-red light during FaRLiP in the marine cyanobacterium Synechococcus sp. PCC 7335. The structure reveals the arrangement of the numerous red-shifted allophycocyanin variants and the probable locations of the chromophores that serve as the terminal emitters in this complex. It also suggests how energy is transferred to the photosystem II complexes produced during FaRLiP. The structure additionally allows comparisons with other previously studied allophycocyanins to gain insights into how phycocyanobilin chromophores can be tuned to absorb far-red light. These studies provide new insights into how far-red light is harvested and utilized during FaRLiP, a widespread cyanobacterial photoacclimation mechanism.

Enhancement of phycocyanin productivity and thermostability from Arthrospira platensis using organic acids.

Intracellular hyperaccumulation of phycocyanin (PC) and its high susceptibility to degradation at higher temperatures are major challenging problems associated with its production from cyanobacteria. The present study evaluated different concentrations of organic acids (1, 2, and 3 mM) (citric acid, acetic acid, succinic acid, fumaric acid, and oxalic acid) under fed-batch mode on the biomass and phycobiliproteins' production from Arthrospira platensis. Besides they were evaluated at 2.5-7.5 mM as preservative to stabilize PC at high temperatures. The incorporation of 3 mM of succinic acid into the cultivation medium enhanced the biomass and PC productivity to 164.05 and 26.70 mg L-1 day-1, which was ~ 2- and threefold higher than control, respectively. The produced PC in this treatment was food-grade with a 2.2 purity ratio. The use of organic acids also enhanced the thermal stability of PC. Citric acid (7.5 mM) markedly promoted the half-life values of PC to 189.44 min compared to 71.84 min in the control. The thermodynamic analysis confirmed higher thermostability of PC in the presence of organic acids and indicated the endothermic and non-spontaneity of the thermal denaturation process. The findings of the present study confirmed that organic acids could be utilized as cost effective and sustainable compounds for promoting not only phycobiliproteins' production but also the thermostability of PC for potential application in food industry.

Recent Progress of Natural and Recombinant Phycobiliproteins as Fluorescent Probes.

Phycobiliproteins (PBPs) are natural water-soluble pigment proteins, which constitute light-collecting antennae, and function in algae photosynthesis, existing in cyanobacteria, red algae, and cryptomonads. They are special pigment-protein complexes in algae with a unique structure and function. According to their spectral properties, PBPs can be mainly divided into three types: allophycocyanin, phycocyanin, and PE. At present, there are two main sources of PBPs: one is natural PBPs extracted from algae and the other way is recombinant PBPs which are produced in engineered microorganisms. The covalent connection between PBP and streptavidin was realized by gene fusion. The bridge cascade reaction not only improved the sensitivity of PBP as a fluorescent probe but also saved the preparation time of the probe, which expands the application range of PBPs as fluorescent probes. In addition to its function as a light-collecting antenna in photosynthesis, PBPs also have the functions of biological detection, ion detection, and fluorescence imaging. Notably, increasing studies have designed novel PBP-based far-red fluorescent proteins, which enable the tracking of gene expression and cell fate.

Molecular dissection of the soluble photosynthetic antenna from the cryptophyte alga Hemiselmis andersenii.

Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αß protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one ß subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein.

Identification of a c-type heme oxygenase and its function during acclimation of cyanobacteria to nitrogen fluctuations.

The mechanisms of acclimating to a nitrogen-fluctuating environment are necessary for the survival of aquatic cyanobacteria in their natural habitats, but our understanding is still far from complete. Here, the synthesis of phycobiliprotein is confirmed to be much earlier than that of photosystem components during recovery from nitrogen chlorosis and an unknown protein Ssr1698 is discovered to be involved in this synthetic process. The unknown protein is further identified as a c-type heme oxygenase (cHO) in tetrapyrrole biosynthetic pathway and catalyzes the opening of heme ring to form biliverdin IXα, which is required for phycobilin production and ensuing phycobiliprotein synthesis. In addition, the cHO-dependent phycobiliprotein is found to be vital for the growth of cyanobacterial cells during chlorosis and regreening through its nitrogen-storage and light-harvesting functions, respectively. Collectively, the cHO expressed preferentially during recovery from nitrogen chlorosis is identified in photosynthetic organisms and the dual function of this enzyme-dependent phycobiliprotein is proposed to be an important mechanism for acclimation of aquatic cyanobacteria to a nitrogen-fluctuating environment.

Microalgae: A Promising Source of Bioactive Phycobiliproteins.

Phycobiliproteins are photosynthetic light-harvesting pigments isolated from microalgae with fluorescent, colorimetric and biological properties, making them a potential commodity in the pharmaceutical, cosmetic and food industries. Hence, improving their metabolic yield is of great interest. In this regard, the present review aimed, first, to provide a detailed and thorough overview of the optimization of culture media elements, as well as various physical parameters, to improve the large-scale manufacturing of such bioactive molecules. The second section of the review offers systematic, deep and detailed data about the current main features of phycobiliproteins. In the ultimate section, the health and nutritional claims related to these bioactive pigments, explaining their noticeable potential for biotechnological uses in various fields, are examined.

Purification and fractionation of phycobiliproteins from Arthrospira platensis and Corallina officinalis with evaluating their biological activities.

Phycobiliproteins (PBPs) are a class of water-soluble pigments with a variety of biological functions that are present in red macroalgae and cyanobacterial species. The crude forms of phycocyanin (C-PC) from the blue green alga Arthrospira platensis and allophycocyanin (APC) from the red macroalga Corallina officinalis were extracted and purified by ammonium sulphate precipitation, anion exchange chromatography, and size exclusion chromatography methods, respectively. The obtained C-PC and APC from A. platensis and C. officinalis were 0.31 mg/mL and 0.08 mg/mL, respectively, with molecular masses of "17.0 KDa and 19.0 KDa" and "15.0 KDa and 17.0 KDa" corresponding to α and ß subunits, respectively. FT-IR was used to characterize the purified APC and C-PC in order to look into their structures. Highly purified extracts (A620/A280 > 4.0) were obtained from subtractions' PC3 and PC4 that were tested for their biological activities. APC and C-PC crude extracts plus their fractions exhibited potent anti-oxidant in different ratios by using three techniques. PC1 showed high anti-inflammatory (75.99 and 74.55%) and anti-arthritic (78.89 and 76.92%) activities for C. officinalis and A. platensis, respectively compared with standard drugs (72.02 and 71.5%). The methanolic and water extracts of both species showed greater antibacterial efficacy against Gram +ve than Gram -ve marine bacteria. Our study shed light on the potential medical uses of C-PC and APC extracted from the tested species as natural substances in a variety of foods and drugs. Further investigations are required to explore the diverse chemical natures of distinct PBPs from different cyanobacteria and red algae because their amino acid sequences vary among different algal species.

Cultivation of blue green algae (Arthrospira platensis Gomont, 1892) in wastewater for biodiesel production.

The production of biodiesel has become an important issue in the effort to reduce gas emissions due to the climate change crisis; therefore, algae have widely used to produce biodiesel for energy sustainability. The present study represented an effort to assess the ability of the alga Arthrospira platensis to produce fatty acids involved in biofuel (diesel) by cultivation in Zarrouk media enriched with different municipal wastewater concentrations. Wastewater was used in different concentrations (5, 15, 25, 35 and 100% [control]). Five fatty acids from the alga were determined and included in the present study. These were inoleic acid, palmitic acid, oleic acid, gamma-linolenic acid, and docosahexaenoic acid. Impact of different cultivation conditions were studied in terms of observed changes in growth rate, doubling time, total carbohydrate, total protein, chlorophyll a, carotenoids, phycocyanin, allophycocyanin, and phycobiliproteins. Results showed an increase in the values of growth rate, total protein content, chlorophyll a, and levels of carotenoids at all treatments except for carbohydrate content, which decreased with an increasing concentration of wastewater. The high value of doubling time (11.605 days) was recorded at treatment 5%. Fatty acids yields were increased at treatment 5% and 15%. The highest concentrations of fatty acids were 3.108 mg/g for oleic acid, gamma-linolenic acid (28.401 mg/g), docosahexaenoic acid (41.707 mg/g), palmitic acid (1.305 mg/g), and linoleic acid (0.296 mg/g). Moreover, the range of phycocyanin (0.017-0.084 mg/l), allophycocyanin (0.023-0.095 mg/l), and phycobiliproteins (0.041-0.180 mg/l) were obtained in treatment with 15-100%, respectively. Cultivation with municipal wastewater reduced the values of nitrate, phosphate, and electrical conductivity as well as increased dissolved oxygen. Maximum electrical conductivity was recorded in untreated wastewater with algae, while the highest level of dissolved oxygen was noted at 35% concentration. The use of the household wastewater is more environmentally friendly as an alternative of the traditional cultivation techniques used for long-term for biofuel production.

The Unique Light-Harvesting System of the Algal Phycobilisome: Structure, Assembly Components, and Functions.

The phycobilisome (PBS) is the major light-harvesting apparatus in cyanobacteria and red algae. It is a large multi-subunit protein complex of several megadaltons that is found on the stromal side of thylakoid membranes in orderly arrays. Chromophore lyases catalyse the thioether bond between apoproteins and phycobilins of PBSs. Depending on the species, composition, spatial assembly, and, especially, the functional tuning of different phycobiliproteins mediated by linker proteins, PBSs can absorb light between 450 and 650 nm, making them efficient and versatile light-harvesting systems. However, basic research and technological innovations are needed, not only to understand their role in photosynthesis but also to realise the potential applications of PBSs. Crucial components including phycobiliproteins, phycobilins, and lyases together make the PBS an efficient light-harvesting system, and these provide a scheme to explore the heterologous synthesis of PBS. Focusing on these topics, this review describes the essential components needed for PBS assembly, the functional basis of PBS photosynthesis, and the applications of phycobiliproteins. Moreover, key technical challenges for heterologous biosynthesis of phycobiliproteins in chassis cells are discussed.

Magnetic Fields as Inducers of Phycobiliprotein Production by Synechococcus elongatus PCC 7942.

This study aimed to analyze the effect of magnetic field (MF) application on the metabolism of Synechococcus elongatus PCC 7942. Concentrations of biomass, carbohydrate, protein, lipid, and photosynthetic pigments (chlorophyll-a, C-phycocyanin, allophycocyanin and phycoerythrin) were determined. In cultures with MF application (30 mT for 24 h d-1), there were increases of 47.5% in total protein content, 87.4% in C-phycocyanin, and 332.8% in allophycocyanin contents, by comparison with the control. Allophycocyanin is the most affected pigment by MF application. Therefore, its biosynthetic route was investigated, and four genes related to its synthesis were found. However, the analysis of the gene expression showed no statistical differences from the control culture, which suggests that induction of such genes may occur soon after MF application with consequent stabilization over time. MF application may be a cost-effective alternative to increase production of compounds of commercial interest by cyanobacteria.

Phycobiliprotein recovery coupled to the tertiary treatment of wastewater in semi-continuous photobioreactors. Tracking contaminants of emerging concern.

This study evaluated a tertiary wastewater treatment technology using cyanobacteria to recover value-added phycobiliproteins. The presence of contaminants of emerging concern (CECs) in wastewater, cyanobacterial biomass and pigments recovered were also analyzed. For this, a wastewater-borne cyanobacterium (Synechocystis sp. R2020) was used to treat secondary effluent from a municipal wastewater treatment plant, with and without nutrients supplementation. Then, the stability of phycobiliprotein production was assessed by operating the photobioreactor in semi-continuous mode. Results showed similar biomass productivity with and without nutrients supplementation (153.5 and 146.7 mg L-1 d-1, respectively). Upon semi-continuous operation, the phycobiliprotein content was stable and reached up to 74.7 mg gDW-1. The phycocyanin purity ratio ranged from 0.5 to 0.8, corresponding to food grade (>0.7). Out of 22 CECs detected in secondary effluent, only 3 were present in the phycobiliprotein extracts. In order to identify applications, prospective research should focus on CECs removal during pigment purification.