Characterization of Pseudomonas strains able to hydrocarbon degradation and polyhydroxyalkanoates production

CARLA DI MARTINO; NANCY I. LOPEZ; LAURA J. RAIGER IUSTMAN

San Miguel de Tucumán, Tucumán

Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO; 2011

Sociedad Argentina de Microbiología General

Bioremediation is an attractive and environmental friendly approach for the cleanup of contaminated sites. It exploits the potential of naturally occurring microbial populations, or in some cases, introduces microorganisms with a known ability to degrade the contaminants. This technique is named bioaugmentation. Desirable characteristics in strains used in bioremediation are the high stress resistance and increased fitness. We propose that those characteristics could be conferred by the polyhydroxyalkanoates (PHA) accumulation capability. Under this context, our approach consisted in to isolate and characterize bacterial strains able to accumulate PHA from hydrocarbon contaminated environments. Two strains - Pseudomonas sp KA-08 and Pseudomonas sp KB-08 - were selected by their ability to grow in high amount of kerosene and diesel and to accumulate PHA from sodium octanoate as well. Comparison of the sequences and phylogenetic analysis of 16S rRNA gene allowed determine that both strains are related to P. putida. PHA was determined by gas chromatography (GC) analysis showing medium-chain length PHA, especially C8. In minimal medium with sodium octanoate as carbon source, KA-08 accumulated 15.3  1.5 % of dry weight and KB-08 7.5  0.8% of dry weight. We studied the type of PHA synthase present on each strain, obtaining positive results for class II phaC2 and phaC1 synthases. The comparison of nucleotide and amino acid sequences of class II synthases in both isolates showed more than 90% of similarity with sequences of Class II polyhydroxyalkanoate synthases from various Pseudomonas strains. In KB-08, a nonsense mutation was observed in phaC2 gene. Both strains were able to grow in crude oil, kerosene, diesel and xylenes as sole carbon source in high agitation conditions (300 rpm). Diesel and kerosene degradation were determined by GC. Kerosene degradation was higher than diesel’s reaching 39% and 35% for KB-08 and KA-08, respectively, after 25 days of culture. No growth in hexane or octane was observed in liquid medium under high agitation condition. However, both strains were able to grow in solid media supplemented with hexane but when octane was used as sole carbon source, only KA-08 was able to grow. PCR analysis of the alkB gene showed differences between the amplicons obtained from KA-08 and KB-08 that could explain these differences. In addittion, both strains showed different results regarding xylenes mix. While KB-08 showed a good growth at 0.5% v/v, KA-08 needed at least 1% v/v to growth, perhaps because a different usage of the xylene isomers. These capabilities of these isolates regarding PHA accumulation and growth in different hydrocarbons and its degradation, will allow us to construct a rational design of bacterial consortia.