Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment u sing either naphthalene, phenanthrene, or biphenyl as the sole source of ca rbon and energy, Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were chara cterized using a variety of phenotypic, morphologic, and molecular properti es, Identification of the isolates based on their fatty acid profiles and p artial 16S rRNA gene sequences assigned them to three main bacterial groups : gram-negative pseudomonads; grampositive, non-spore-forming nocardioforms ; and the gram-positive, spore-forming group, Paenibacillus, Genomic digest patterns of all isolates were used to determine unique isolates, and repre sentatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas y anoikuyae B1, and Mycobacterium sp, strain PY01, None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform iso lates showed homology to the PY01 genes, and only members of the pseudomona d group showed homology to the NCIB 9816-4 or GZ42 probes, The Paenibacillu s isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the th ree groups showed that the phenanthrene-enriched isolates are able to utili ze a greater number of PAHs than are the naphthalene-enriched isolates. Ino culating two of the gram-positive isolates to a marine sediment slurry spik ed with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to th e two- and three-ringed PAHs and biphenyl, This study indicates that the rh izosphere of salt marsh plants contains a diverse population of PAM-degradi ng bacteria, and the use of plant-associated microorganisms has the potenti al for bioremediation of contaminated sediments.