PRODUCTION; EXTRACTION AND PURIFICATION OF PHENAZINES PRODUCED BY Pseudomonas aeruginosa PAO1

Phenazines are heterocyclic aromatic nitrogen compounds with carboxy and hydroxy substituted derivatives that exhibit antimicrobial activity. These compounds may have various shades and are produced under the control of the mechanism of quorum sensing by various genera of bacteria. Phenazine compounds produced by Pseudomonas aeruginosa have varied biological functions and are important for microbial competitiveness and virulence in human and animal hosts. On other hand, there are reports in the scientific literature suggesting potential antioxidant and non-cytotoxic properties of these compounds, indicating a great potential of application in the pharmaceutical and cosmetic industries. The objective of this study was to develop a process of production, extraction and purification of phenazines produced by Pseudomonas aeruginosa PAO1. Submerged cultures were carried out in Erlenmeyer flasks of 250 ml containing 100 ml of salt medium (containing per liter: 3.0 g KH2PO4, 7.0 g K2HPO4, 0.2 g MgSO4.7H2O and 1.0 g (NH4)2SO4) added of glycerol (3% v/v), which were incubated at 37°C with shaking at 200 r.p.m. for 12 days. Extraction was performed from the cell-free supernatants of cultures with chloroform (1:1 v/v). The aqueous phase was acidified (pH 4.5) and centrifuged with Celite 545 and activated charcoal, and then, washed with water, followed by methanol 50 % (aqueous solution) and eluted with pyridine 5 % (aqueous solution). The organic phase was subjected to a silica column and eluted sequentially with dichloromethane, chloroform, chloroform: methanol (1:1) and methanol: 2% acetic acid. Finally, the pigment samples obtained were analyzed by thin layer chromatography (TLC). The production conditions employed were efficient for the synthesis of 4 different pigments by Pseudomonas aeruginosa PAO1, and the shades obtained were green, blue, yellow and red. The water-soluble fraction showed a brownish coloration after extraction and after the purification process became red. The soluble fraction in chloroform was green and after purification on silica column with different solvents four different fractions were obtained based on polarity differences: yellow, green, blue and colorless. TLC showed that the purification and separation process of the phenazines was effective and it was possible to detect them through the migration of the bands. Thus, the results showed that it is possible to isolate phenazine pigments from Pseudomonas aeruginosa, enabling to test its antibiotic, antifungal and antitumor activities. Therefore, these molecules may be of great interest for formulations of beneficial products for the treatment of various diseases and in the cosmetics and pharmaceutical industry.