Green Chemistry PAPER Cite this: Green Chem., 2016, 18, 4287 Received 14th April 2016, Accepted 28th April 2016 DOI: 10.1039/c6gc01059h www.rsc.org/greenchem Recovery of phycobiliproteins from the red macroalga Gracilaria sp. using ionic liquid aqueous solutions† Margarida Martins, a Flávia A. Vieira, a Isabel Correia, b Rute A. S. Ferreira, c Helena Abreu, d João A. P. Coutinho a and Sónia P. M. Ventura* a Bioactive compounds extracted from natural renewable sources have attracted increased interest from both industry and academia. Several biocompounds are present in red macroalgae, among which R-phycoerythrin (R-PE), which is a phycobiliprotein with a wide range of applications. The major draw- back associated with it is the absence of an efficient, low cost and green extraction and purification methodology capable of recovering phycobiliproteins (and, in particular, R-phycoerythrin) from the biomass, while maintaining their structure and activity. The search for novel and higher performance extraction processes is thus of extreme relevance. In this work, aqueous solutions of ionic liquids were screened for the extraction of phycobiliproteins from Gracilaria sp. The most promising solvents were identified and operational conditions such as extraction time, solid–liquid ratio, solvent concen- tration and pH were optimized aiming to develop a new and more efficient approach to extract phycobili- proteins. The efficiency of the proposed process is demonstrated with aqueous solutions of cholinium chloride, since the extraction of phycobiliproteins was increased to 46.5% when compared with the con- ventional methodology, while the protein secondary structure and the chromophore conformation integrity are maintained. Introduction Phycobiliproteins are the main photosynthetic pigments present in red algae, cyanobacteria, and cryptomonads, a uni- cellular eukaryotic alga. 1 These are light-harvesting pigment– protein complexes organized in vivo in supramolecular struc- tures called phycobilisomes, located at the stroma, on the external structure of the thylakoid membrane. 2 The phycobili- proteins allow simultaneously the transfer of light energy to the living organism and their survival at low light intensities. 2 Due to their high solubility in water, stability, 3,4 and bioactiv- ity, phycobiliproteins 5 have gained special significance in many different sectors, such as the food, 6 pharmaceutical, and cosmetic industries. 2,7 Among the phycobiliproteins, R-phy- coerythrin has been singled out as an important tool in the field of medical diagnosis, and biomedical research, 8 due to its excellent optical and spectroscopic properties, high absorp- tion coefficient, and high fluorescence yield. These fluorescent pigments have also shown antioxidant and antitumoral activi- ties. 9 More recently, phycobiliproteins have called attention by their potential application in the energy field, 10 namely in the production of “dye-sensitized solar cells” (DSSCs). DSSCs are non-tracking concentrators that redirect solar radiation into simple slab waveguides to be collected by a photovoltaic cell mounted at the edge of the slab. 10 Globally, there are about 50 producers of phycobiliproteins, with their market prices ranging from $6 to $12 per mg. 11 Due to their high cost, the market is still small, although with estimated yearly growths of 20%. 11 Taking into account the potential of phycobiliproteins and their range of applications, the development of novel efficient processes to extract and purify these fluorescent pro- teins from the fresh biomass is of utmost importance, albeit a challenging task. The common extraction practices currently used are based on solid–liquid extraction with buffer aqueous solutions, namely sodium phosphate, 12,13 followed by chrom- atography and gel filtration, 13,14 or enzymatic processes 1,15 to purify R-phycoerythrin from the phycobiliproteins. While some studies describe the use of different solvents, including the buffered solutions and water to extract the phycobilipro- teins [R-phycoerythrin results found between 0.666 mg g -1 (for Gracilaria verrucosa 16 ) and 1.73 mg g -1 (for Gracilaria lemanei- † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c6gc01059h a CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail: spventura@ua.pt b Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal c CICECO - Aveiro Institute of Materials, Department of Physics, Universityof Aveiro, 3810-193 Aveiro, Portugal d ALGAplus® Ltda, Travessa Alexandre da Conceição, 3830-196 Ílhavo, Portugal This journal is © The Royal Society of Chemistry 2016 Green Chem. , 2016, 18, 4287–4296 | 4287 Published on 02 May 2016. Downloaded by Universidade de Lisboa on 18/10/2016 11:15:11. View Article Online View Journal | View Issue