Phenanthrene degradation by Halobacterium piscisalsi: Design and optimization of a method for biofilm cultivation in continuous flow system

DI MEGLIO, LEONARDO; BUSALMEN, JUAN PABLO; NERCESSIAN, DÉBORA

Córdoba

Congreso; XI Congreso de la Sociedad Argentina de Microbiología General (SAMIGE); 2015

Phenanthrene is a three ring polycyclic aromatic hydrocarbon (PAH) used as a PAH model to study aerobic degradation. PHAs are highly toxic because of their irritant, mutagenic and carcinogenic effects on living organisms. One important source of PHAs pollution is the produced water of oil production process, which is indeed generated in large amount. Bioremediation has become one of the most effective and economical strategy to remove these contaminants. This technology employs microorganisms which are able to metabolize toxic compounds and transform them into harmless ones. In this direction, much has been studied about bacterial remediation, but the knowledge about metabolic pathways and enzymes involved in hydrocarbon degradation in hypersaline environments is scarce. Because of the high salinity of produced water, microorganisms conventionally employed in PHAs remediation cannot be effective in biological treatment of this wastewater, which tookresearchers to consider extremophilic microorganisms, as halophilic archaea. Firstly, we identified which of the microorganisms isolated from La Pampa saltern ponds (Argentina) were able to degrade phenanthrene in planktonic culture. These assays were performed in liquid medium under low oxygen concentration in the presence of 0.02% phenanthrene, for four weeks. Degradation products and the remaining phenanthrene were extracted from the extracellular medium with ethyl acetate and analyzed by High Resolution Liquid  Chromatography (HPLC).  Halobacterium piscisalsiwas the microorganisms selected toperform biofilm assays due to its capacity to completely degrade phenanthrene and its higher growth rate than the other isolates. Biofilm assays were performed in a continuous flow growth chamber, using glass coverslips as substrate for the sessile population. With this system, we could study microbial activity  in situthrough direct microscopic observation. For the optimization and tune up of the biofilm system, different strategies were conducted in order to obtain phenanthrene derivatized cover slips. Time of cell adhesion (before starting the continuous flow) was adjusted to achieve cell attachment to thehydrophobic surface. Flow rate and temperature assay were also adjusted to avoid crystals dissolution. Results suggest that H. piscisalsiis able to develop biofilms on phenanthrene derivatized substrate and to grow using it as its sole carbon source. Phenanthrene crystals degradation through time was observed and the association between cell and phenanthrene crystals was determined by scanning electronic microscopy (SEM). As phenanthrene emits in the blue range when excited with UV, we are optimizing a quantification methodology to follow fluorescence along time. To achieve this, we are changing different parameters like microscopic magnification and UV exposition, aiming to develop a phenanthrene  biodegradation system not described before.