Study of Pseudomonas strains able to degrade hydrocarbon and the role of polyhydroxyalkanoates in biosurfactant production

CARLA DI MARTINO; ANTONELA COLONNELLA; NANCY I. LOPEZ; LAURA J, RAIGER-IUSTMAN

Rosario, Santa Fe

Congreso; X Congreso Argentino de Microbiología General; 2013

SAMIGE

Bioremediation is an environmental friendly approach for the cleanup of contaminated sites. In some cases, seeding of microorganisms with a known ability to degrade contaminants (bioaugmentation) is an option. In this case, strains that show high stress resistance and increased fitness are desirable. Pseudomonas sp KA-08 was selected by its ability to grow in high amount of kerosene, diesel and xylene as sole carbon source, to accumulate polyhydroxyalkanoates (PHA) and synthesize biosurfactants. Those characteristics made Pseudomonas sp KA-08 an interesting candidate as bioaugmentation agent. Xylene degradation was assayed in minimal media supplemented with 1% xylene or microcosms constructed with natural freshwater from Rio de la Plata. The remaining xylene after 15 days was measured by head space GC, reaching a 70% of degradation in both, culture and microcosms conditions. Biosurfactant production was analyzed by TLC and CTAB methylene blue agar plates revealing the presence of glycolipids and/or anionic moieties. As PHAs were postulated to enhance stress resistance, a PHA- mutant of Pseudomonas sp KA-08 was constructed by replacing phaC1, phaZ and phaC2 genes for a kanamycin resistance cassette (Km) by CrossOver PCR deletion. This mutation did not affect growth capabilities in PHA accumulating conditions (ME + sodium octanoate) since the CFU/ml counts were similar. To analize the effect of PHA accumulation capability in biosurfactant production, surface tension (ST), emulsification activity (E24) and hydrophobicity index (HI) were assayed. Reduction in the ST was always superior in WT strain than in the mutant when growing in PHA accumulating conditions. E24 revealed a high emulsification activity, near to 60% but the capability to accumulate PHA seems to have no effect in the emulsion stability generated by the secreted surfactants. HI assay showed that there is no difference in the membrane hydrophobicity from WT or PHA- mutant when they grew in PHA accumulating contidion. Interestingly, when xylene was used as HI hydrocarbon added into the cell suspensions, cell recruitment in this organic phase was allways higher than those observed when using other hydrocarbon as diesel. This could be an evidence of a membrane adaptation to aromatic hydrocarbon polluted environments. As BTEX contaminated waters are also often associated with heavy metal contamination, preliminary studies of metals (copper (Cu (II)) resistance were performed ussing Pseudomonas sp KA-08, Pseudomonas extremaustralis and their PHA- mutants. All the assayed strains showed high (more than 3 mM) resistance to Cu (II). In conclusion, Pseudomonas sp KA-08 seems to be a good candidate as biorremediation agent for xylene contaminated freshwater. PHA accumulation seems to increase the production of tensoactive compounds without affecting bacterial growth.