Formulation of low-cost culture media for the immobilization of Pseudomonas sp. P26 on natural supports

Abstract

Pseudomonas sp. P26 is an environmental bacterium with desirable physiological and safety properties for environmental bioremediation processes. The objective of this study was to formulate low-cost culture media using industrial by-products for the immobilization of Pseudomonas sp. P26 (P26) on natural organic supports. The bacterial strain P26 was immobilized on peanut shells and walnut shells using a standard culture medium (LBm) and media formulated with industrial by-products (corn steep water –CSW– and crude glycerol) and K2HPO4 (as an inorganic biostimulant). The metabolic activity of the immobilized bacteria was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. In the selected immobilized system, the number of viable cells and intracellular inorganic polyphosphate (polyP) content were quantified (using a spectrophotometric method to measure inorganic phosphate released by the acid hydrolysis of polyP), and electron microscopy analyses were performed. In the first trial, significantly higher metabolic activity values were observed in walnut shells compared to peanut shells, in the control medium LBm, both in the presence and absence of inorganic phosphate. In the second trial, the highest metabolic activity values were determined in the medium named M4 (formulated with 2.5% v/v CSW + 1% v/v glycerol) and in LBm, with and without inorganic phosphate. In media with added inorganic phosphate, no significantly higher metabolic activity values were observed compared to those without this substrate. Electron microscopy revealed biofilm formation on the walnut shells, few connections between bacterial cells, and moderate extracellular material production. PolyP accumulation under immobilized conditions was higher than in planktonic cultures. An immobilization system based on Pseudomonas sp. P26 cultivated in corn steep water and crude glycerol, using ground walnut shells as support, was developed. The immobilized system is a potential candidate for future studies on the removal of aromatic compounds from petroleum.