Diversity of polycyclic aromatic hydrocarbons-degrading bacteria in intertidal marine sediments of Patagonia

RIVA MERCADAL, J.P.; LOZADA, M.; DIONISI, H.; SIÑERIZ, F.; FERRERO, M.A.

Dalian, China

Simposio; The thirteenth International Biotechnology Symposium and Exhibition (IBS2008); 2008

International Union of Pure and Applied Chemistry (IUPAC)

The diversity of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria present in the marine environment remains largely unknown, in particular in sub-antartic regions, the presence of PAHs in coastal marine sediments of Patagonia can be found as a result of anthropogenic activities or due to natural events such as petroleum seeps. In this study, seven different intertidal marine sediments along patagonian coast of Argentina were analyzed on their intrinsic capability of degrading PAHs. Enrichment cultures were set up for 15 days at 20 ºC in minimal medium supplemented with naphthalene or phenantrene as sole carbon and energy source using coastal sediment as inoculum. Total DNA of each naphthalene or phenantrene-enriched bacterial population was extracted by using the CTAB method. All of the 14 samples were analyzed by a combination of PCR-DGGE using universal primers 341GC and 518R for eubacterial 16S rDNA [1]. Band profiles were compared between several culture conditions and sampling sites, considering band richness and diversity. Nearly two hundred bands were reamplified and sequenced in orden to determine the phylogenetic status of the different bacterial populations. Approximately 25 % of bands recovered from the gels resulted in operational taxonomic units (OTUs) with 100 % identity to Cycloclasticus sp., a versatile PAH-degrading bacteria not reported at these coasts and nearly 10 % of sequenced bands showed high identity with Shewanella sp. Other OTUs related to PAH-degrading bacteria found between recovery bands belonged to genus Vibrio, Pseudoalteromonas, Pseudomonas, Photobacterium and Rhodobacter. DNA samples were also used as template for amplification of internal fragments of some alpha subunit of iron sulfur protein (ISP) of initial PAH-dioxygenase genes. Primers cyc-p 372f and cyc-p 854r [2], and primers nahAc 149F and nahAc 1014R [3] designed with GC clamp for DGGE were used for these assays. The presence of phnA1-like genes of Cycloclasticus was observed in five of seven sediments analyzed and nah-like genes of Pseudomonas or Sphingomonas in the others. Only one band was obtained of nah-like gene, but several bands were observed at bacterial populations amplified for phnA1-like genes. All bands were recovered from DGGE gel and reamplified for further sequencing. Amplification products identified as nah-like genes reveal the potential intrinsic capability of degrading PAHs, nevertheless, presence of important amount of OTUs related to the genus Cycloclasticus and their characteristic type phnA1 gene in enrichment cultures from different sites along Argentinean Patagonia coast suggest that it could play an important role in the marine degradation of PAHs as it was demonstrated by studies in several sites of the northern hemisphere [4]. References [1] Muyzer, G., de Waal, E.C., Uitterlinden, A.G. (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 59 (3): 695-700 [2] Lozada, M., Riva Mercadal, J.P., Guerrero L.D., Di Marzio, W.D., Ferrero, M.A., and Dionisi, H.M. (2008) Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia BMC Microbiology. 8:50 [3] Ferrero, M., Llobet-Brossa, E., Lalucat, J., Garcia-Valdes, E., Rossello-Mora, R., Bosch, R. (2002) Coexistence of two distinct copies of naphthalene degradation genes in Pseudomonas strains isolated from the western Mediterranean region. Appl Environ Microbiol.  68:957-962. [4] Kasai, Y., Kishira, H., and Harayama, S. (2002). Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. Appl. Environ. Microbiol. 68:5625-5633.