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SYMPOSIUM on Marine Accidental Oil Spills
Degradation of the PAHs present in the Prestige fuel oil by a
microbial consortium from Corrubedo (Galicia). Community
structure of the pyrene-degrading populations
Joaquim VILA, Lida ARIAS, Sara GALLEGO, José Maria NIETO
and Magdalena GRIFOLL
Departament de Microbiologia, Universitat de Barcelona, Diagonal 645,
08028-Barcelona, Spain
ABSTRACT
Our research focuses on the microbial processes involved in the degradation of high molecular
weight (HMW) PAHs in the environment, using pyrene and fluoranthene as model compounds.
Enrichment procedures for pyrene and fluoranthene degrading strains from contaminated soils
often leads to the isolation of actinobacteria, belonging to the genus Mycobacterium. However,
little is known about the microbial populations involved in HMW-PAHs in marine environments.
After the Prestige oil spill a bioremediation study was performed in the beach of Sorrizo (A
Coruña). One of the objectives of this study was the development of an autochthonous
microbial consortium (UBF) able to degrade the Prestige fuel oil for field applications. This
consortium has been maintained for more than three years by successive transfers (15 days).
The objective of the present work is to evaluate the HMW PAH-degradative capabilities of
consortium UBF, describe the processes involved, and characterize the microbial populations
carrying them.
Cultures of the microbial consortium UBF in artificial sea water and fuel degraded 73% of the
aliphatic fraction in 15 days, while the aromatics where reduced in a 37%. GC-MS SIM analyses
of the residual TPHs showed a complete depletion of all the PAHs with 2 and 3 rings, together
with fluoranthene and benzo(a)anthracene. Pyrene, chrysene and benzo(a)pyrene were also
extensively attacked (75%, 70% and 20%, respectively).
Given this high degrading potential towards PAHs and the lack of knowledge about the
microbial populations involved in PAH degradation in marine environments, a study of the PAH
degrading populations present in the consortium UBF was carried out. Attempts of isolation of
PAH degrading strains by classical enrichment and plating methods were unsuccessful,
probably due to the complex community interactions between strains. Therefore, a study based
in culture-independent methods was initiated. To study the microbial populations involved in the
degradation of pyrene and fluoranthene, the microbial consortium UBF was used as inoculum
for two enrichment subcultures in sea water and fluoranthene or pyrene as sole carbon sources.
After several transfers and confirmation of substrate removal, the total DNA of the new
consortia was obtained, the 16S rDNA was amplified by PCR, and a clone library was
developed. For the PCR amplification we used universal primers, while the PCR amplification
products were cloned using the p-GEM-T Easy vector, and transformed into E.coli JM-109 to
obtain a clone library formed by 150 positive clones for each consortia. The cloned 16S rDNA
fragments of 40 of these positive clones were amplified by PCR using primers from the p-GEMT Easy vector, and the amplification products were analyzed by RFLP using the frequent-cutting
restriction enzyme Msp I to avoid sequence redundancy. The different ribotypes were grouped
according to their unique pattern and one fragment of each group was sequenced. Based on
the sequence analyses the microbial structure of the pyrene degrading consortia was
established.
Since FISH analyses of the microbial consortia UBF using specific DNA probes for
Mycobacterium showed the presence of members of this group in the mixed culture, a new
clone library, obtained by PCR amplification of the 16S rDNA using specific primers for
Mycobacterium, is now being developed.
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