Isolamento e caracterização de micro-organismos produtores de polihidroxialcanoatos (PHA)

Abstract

Polyhydroxyalkanoates are biodegradable polyesters from microbial origin composed of monomers of hydroxyalkanoic acids and/or hydroxyalkenoic ones. They are synthesized intracellularly and deposited as granules in bacterial cultures in the presence of carbon source in excess and a limiting nutrient growth, whose function is to stock carbon and energy for microorganisms. Among the main factors that affect economic viability of PHA production is the selection of bacterial strains that are efficient in the conversion of substrates into the biopolymer, with production above 40% of the dry cell weight in the low-cost medium growth. Through these considerations, the objective of this work was to isolate, select, identify and characterize new microorganisms’ strains with potential polyhydroxyalkanoates (PHA) production from hydrocarbon contaminated environments. Samples of soil and water were collected (all contaminated with hydrocarbons) in the regions of Port of Itapóa - SC, Morretes - PR, Antonina - PR, Vila da Glória - SC and of the sand of Future Beach in Fortaleza – CE. Insulation techniques have been applied to the surface of pure colonies, in Medium Mineral Saline without limitation. The selection of microorganisms with PHA production capacity occurred after the sowing in Medium Mineral Saline with limited addiction of Nile Red, following the inspection of bacterial colonies in the transilluminator. The cell extraction of the PHA occurred with the aid of sodium hypochlorite, acetone and alcohol. The microorganisms were characterized by its morphology, motility, spore production, Gram stain, growing in anaerobiosis, oxidase test, catalase, cytochrome oxidase, citrate, acetoin, acid production from sugars (glycose, fructose, mannitol, maltose and xylose), nitrate and starch hydrolysis, gelatin, casein and urea. The identification of microorganisms was by molecular methods. There were 32 isolated colonies and 4 selected microorganisms with PHA production capacity, two genders were identified, the Sphingomonas sp. and Arthrobacter sp.. The biochemical characteristics of the Sphingomonas sp., with the largest PHA production (32,8%), are: gram-negative, motility in semi-solid medium, negative for spore production, fructose fermentation, glucose mannitol and xylose does not ferment (does not produce butylene glycol), citrate use, fermentation of a mixed acid type, hydrolysation of casein and urea, but does not hydrolyses starch and gelatin. It also grows anaerobically at 20ºC and does reduce nitrate. The analysis in Fourier Transform Infrared Spectroscopy (FTIR) of the biomaterial extracted from the microorganism Sphingomonas sp., showed in its characteristics, bands of the presence of P(3HB). However, there is need to optimize the cultivation media with the aim to stimulate biopolymer production.