# Extraction of Pigments from Microalgae and Cyanobacteria--A Review on Current Methodologies
## 2 Classic Extraction  
Classic extraction of microalgae and cyanobacteria pigments is performed using a solvent extraction associated, or not, to a thermal treatment (heat or cold), as depicted in Figure 1. The process of solvent extraction entails mixing the two phases (solvent + biomass) so that the solute can come into contact with the solvent until an equilibrium is reached. The transference from one phase to the other is driven by chemical affinities. The solution of solvent plus solute obtained at the end of the process is called \"extract\". The main advantage of this kind of extraction is the reduced cost in terms of infrastructure and operating procedures. Solvent extraction, on the other hand, frequently demands large volumes of organic solvents for carotenoids extraction, or even a lengthy processing time, as in the case of phycobiliproteins extraction, due to cyclic thermal treatment. In both cases, efficiency is usually not enough for industrial application [12]. When selecting a solvent, solubility of the compound, toxicity, and ecological impact of residues are important points to consider. The extraction performed with organic solvents happens when the solvent is absorbed within the cell wall, causing some rupture and making the cell content available for extraction. The complete extraction can take a long time and usually improved efficiency can be achieved with stirring or mixing [12; 13]. Only in the few cases where microcalga does not require harsh disruption methods due to cell composition can solvent extraction be as efficient as other methods. For example, _Isachrysis galbana_ is a source of fucoxanthin, which can be obtained via a single solvent extraction with ethanol [14]. Classical extraction of astaxanthin from _H. pluvialis_ is performed by using organic solvents such as chloroform and methanol or more eco-friendly solvents such as acetone, ethyl acetate, and ethanol. The single solvent extraction with acetone can induce cell-wall disintegration and extraction of pigments, although the efficiency is relatively low and the process requires a long time [15; 16]. Thus, it is possible to use a mixture of solvents or extraction steps in order to increase efficiency. [PERSON] et al. [17] used both ethanol and ethyl acetate in a 1:1 mixture for the extraction of astaxanthin from _H. pluvialis_, the optimization was performed varying the percentage of ethanol from 30% to 70%, and consequently changing the polarity of the solvent mixture. Furthermore, a simple solution for enhancing the extraction rate is to apply a thermal treatment. Carotenoid's extraction can be more efficient if the process is performed at a temperature between 50 to 65 \({}^{\circ}\)C. At these temperatures, the cell wall becomes less resistant, and the process is further enhanced by the increased solubility of pigments in organic solvents at higher temperatures [18]. However, a long exposure to temperatures above 70 \({}^{\circ}\)C can cause degradation of carotenoids and chlorophylls that are considered thermally sensitive compounds [19]. When it comes to phycobiliproteins, one of the most common extraction techniques uses the freeze-thaw method, using phosphate buffer or water as a solvent. The freeze-thaw method consists of the crystallization of the intracellular water by freezing the wet biomass, followed by thawing under refrigeration temperatures (4 \({}^{\circ}\)C), which causes cell lysis due to the expansion of the ice crystals. The extraction can involve several freeze andthaw cycles, leading to a time-consuming process and lack of reproducibility, which is highly affected by the number of cycles [20; 21]. Optimization of the process involves adjustment of solvent, freezing temperature, freezing and thawing time, and biomass-to-solvent ratio, among other factors. The extraction of phycocyanin is not limited by low temperature; [PERSON] et al. [22] optimized _A.