*4.4. Extraction Approach*

Another phase important in yield improvement consists of the extraction step, which helps in concentration and pre-purification of fungal pigments. Prior to the extraction, it is necessary to take into consideration where the pigments produced are deposited. Classically, the extraction procedure is usually accomplished by liquid-liquid extraction of the broth with medium polarity solvents such as ethyl acetate. This extraction works well to obtain extrolytes, i.e., extracellular metabolites present in the broth or linked to the external surface of fungal biomass. On some occasions, mycelial adhesion is verified, as reported for a water-soluble extracellular yellow pigment produced by a *Monascus* in submerged fermentation. This effect was reversed furnishing sodium and potassium nitrate as nitrogen source to the fungus. Sodium nitrate is suggested to reduce the total amount of extracellular polysaccharides, increase extracellular proteins, and diminish the viscosity of the fermentation broth, rising pigment recovery [16].

Although effective for extraction of metabolites produced in liquid cultures, ethyl acetate is not a choice solvent in terms of toxicity. Non-toxic and easily available ethanol is a better choice for the extraction step, although can only be applied to solid state fermentation, as it is water-miscible and cannot be utilized to extract aqueous liquid media. Ethanol was utilized for pigments extraction in the solid-state fermentation of *M. purpureus* M9 using durian seed as substrate. Extractions were carried out at two temperatures (30 and 60 oC) using a mixture of ethanol and water in different proportions (10:0; 9:1; 8:2; 7:3; 6:4 and 5:5). The most effective conditions for pigments recovery were achieved using the lowest ethanol:water ratios at 30 ◦C [105].

Occasionally the pigments remain inside the cells requiring disintegration and dissolution of the glucan-chitin complex of the wall cell to be recovered, therefore demanding alternative extraction procedures [106], while cellular lysis is necessary for recovering intracellular metabolites. In this way, *T. amestolkiae* DPUA 1275 was subjected to an alternative extraction procedure to recover red pigments. The procedure was conducted with aqueous solutions of imidazolium salt instead of organic solvents, together with ultrasound-temperature-assisted mechanical cell disruption to enhance the recovery of intracellular *T. amestolkiae* pigments [103].

Cell Pressurized Liquid Extraction technique was utilized to recover pigments produced by mycelial biomass of *Talaromyces* sp. 30570 (CBS 206.89 B) isolated from the coral reef of the Réunion island (France) and cultivated in PDB media containing complex organic nitrogen sources like amino acids and proteins. Eco-friendly solvents were chosen for the extraction (90 ◦C and 10 MPa) as water, methanol and/or ethanol. At the end, twelve nitrogen-containing azaphilone red pigments were identified while known mycotoxins

were not produced [13]. Two-phase aqueous extraction [107] was successfully tested for the extraction of pigments from *T. albobiverticillius*. These organic solvents free techniques guarantee good extraction yields without structural damage in the extracted compounds. Cell disruption methods for improved extraction of pigments from microorganisms were recently reviewed [108].

For pigments production, submerged fermentation is preferable, as it produces better yields, has lower risk of contamination and is easier to monitor when compared to cultivation in solid medium [109]. In addition, using submerged fermentation, it is possible to separate intracellular and extracellular pigments, soluble in the culture medium [102]. However, it is known that not all species of pigment-producing fungi have the ability to diffuse these pigments into the culture medium [73]. Among the techniques to increase the production of extracellular pigments, the design of mutant strains of *M. purpureus* [102], the addition of glycerol to the cultivation medium of *M. pilosus* MS-1 [110] and the establishment of a hyperosmotic environment to *M. ruber* CGMCC 10910 [82] were successfully utilized. The last two methods are related to the regulation of metabolism and gene expression caused by environmental stress.
