Study of Pseudomonas sp. 14-3 capability to accumulate polyhydroxybutyrate (PHB) from different hydrocarbon sources

CARLA DI MARTINO; PETER DÖHMER PISANI; JIMENA A. RUIZ; NANCY I. LOPEZ; LAURA J. RAIGER- IUSTMAN

Rosario- Argentina

Congreso; V Congreso Argentino de Microbiología General (SAMIGE); 2008

Sociedad Argentina de Microbiología General

Pseudomonas sp. 14-3 strain was isolated from the Antarctic environment. It´s able to grow at low temperatures and has a high stress resistance. In conditions of Carbon-Nitrogen unbalance, this strain produces a large amount of PHB (70 to 80% PHB / dry weight). Due to its characteristics, this strain could be a good candidate for the remediation of environments contaminated with hydrocarbons, which have a high C/N ratio. We challenged this strain with several hydrocarbons without success. For that reason, Pseudomonas sp. 14-3 and its mutant, Pseudomonas sp.14-3∆phb, were transformed with pGEc47 plasmid, in a way to provide the necessary genes for alkane degradation (alkBAC-alkR).Pseudomonas sp.14-3∆phb is a phaC mutant (PHB synthase) and is unable to synthesize PHB, but it is able to synthesize PHA. To analyze if the strains were able to grow in the presence of hydrocarbons, the cultures were grown at 32°C in 100 ml E2 minimal medium supplemented with 10% w/w of n-octane, gasoline, kerosene and diesel as the only carbon source. To avoid solvent evaporation, the cultures were done in 500 ml capped bottles in strong agitation (300 rpm). The growing rate was analyzed by OD measurement during 10 days. Alternatively, the strains were grown in Petri dishes with E2 agar supplemented with the hydrocarbons cited above. To analyze PHB or PHA accumulation, sodium octanoate was utilized as sole carbon souse as positive control and a sample was taken even 48 hr. PHB accumulation was detected by staining with Nile Blue dye and observed in a fluorescence microscope. Pseudomonas sp. 14-3 and its mutant, Pseudomonas sp.14-3∆phb, were able to grow in E2 minimal media supplemented with n-octane, kerosene and diesel as the only carbon source. Neither the wild type nor the mutant were able to grow in a liquid media supplemented with gasoline as sole carbon source, but in E2 gasoline Petri dishes, colonies appeared after six days, perhaps due the presence of bacterial growth inhibiting substances in the gasoline. For all tested hydrocarbons, the wild type strain always showed a higher OD than the PHB-. The staining with Nile blue revealed that the wild type strain accumulated PHB after the forth day of culture when hydrocarbons were used as the only carbon sources, while in the presence of sodium octanoate, the PHB accumulation was observed after 16 hours. Neither PHB nor PHA accumulation was observed in Pseudomonas sp.14-3∆phb strain. These preliminary studies pretend to be the first step to analyze the effect of the capability to accumulate and degrade PHB into the hydrocarbon bioremediation.