Produção de Ficocianina de Nostoc sp. e Anabena variabilis e recuperação por sistemas aquosos bifásicos

Abstract

Cyanobacteria are organisms capable of producing a diversity of biocomposites with high added value, including phycobiliproteins (PBP), which are pigments used as natural dyes for food and cosmetic products, as well as molecular fluorescent markers. This work aimed to study the process of production and recovery of PBP from cyanobacteria, Nostoc sp. and Anabaena variabilis. The only PBP detected in the extract of both strains was phycocyanin (PC). The medium of cyanobacteria was modified to evaluate the effect of the concentration of carbon source (glucose) and nitrogen source (sodium nitrate) and the effect of color (white, yellow, blue, green and red) on biomass growth and PC synthesis. For the PC recovery stage, the aqueous two-phase system (ATPS) was chosen, as an attractive technique for biomolecules, as it presents a large amount of water in both phases, providing a non-toxic and compatible environment. In this sense, the effect of adding NaCl to ATPS of PEG-potassium phosphate on PC recovery was evaluated. Furthermore, it was also tested ATPS using tri-block copolymers of different chains of PEO (poly (ethylene oxide)) (F68, L62 and L64) and the type of salt (sodium citrate and potassium phosphate) in different proportions, to test the increase in PC recovery and initial purity. Copolymers are thermo-sensitive and can be recovered and reused by heating, different from PEG. Thus, recovery was also evaluated by terms of separation of the copolymers used. In respect to the effect of cultivation parameters, the best result was found in supplementing the medium for the Nostoc sp. of 2 g.L-1 of glucose and 5 mM of sodium nitrate in a combined form, obtaining PC concentrations of 133.03 mg.g-1 and cell concentration of 0.53 g.L-1. Regarding the effect of the color of light, in general, using white light allowed the highest cell growth and when combined with the supplementation assay, it presented about 0.43 gL-1 for both strains studied after 14 days of cultivation. For the production of PC, under supplementation of the medium, the red light showed better response for both strains, equivalent to 141.97 mg.g-1 for Nostoc sp. and 161.06 mg.g-1 for A. variabilis. The study of PC recovery by ATPS, indicated the positive effect of “salting-out” with the addition of sodium chloride (NaCl), with 6% (w/w) being the best condition for Nostoc sp., Reaching purity (P) of 1,13, recovery (R) of 84.5% and partition coefficient (K) equal to 9.46. As for A. variabilis, the best conditions were for adding 6 and 8% (w/w), reaching (P) 1.16, (R) 83.35% and (K) 9.80. For ATPS of the copolymer-salt type, and 25% copolymer and 8% salt ratio, L64 + potassium phosphate was the best system for the recovery of PC from the extracts of the two strains, with the protein partitioning occurring in the bottom phase, reaching (R) equivalent to 83.40% for Nostoc sp. and 86.76% for A. variabilis. In the second proportion, 15% copolymer and 12% salt, the PC migrated to the top phase, with the F68 + potassium phosphate system providing a better result for both strains, reaching (R) of 86.46% for Nostoc sp. and 86.83% for A. variabilis. In the step of recovering the copolymer by thermal separation, it was possible to recover L62 (94.50%) and L64 (96.54%). For the F68 copolymer, there was no phase separation in the tested temperature range. The study showed that higher levels of phycocyanin can be obtained by modifying the medium of cyanobacteria, especially with the supplementation of carbon and nitrogen sources, and that the recovery of this pigment is viable by ATPS, both for PEG and copolymer media.