Download Hydrocarbon-degrading marine bacteria relevant in the nitrogen cycle

VERTIMAR-2005
Symposium on Marine Accidental Oil Spills
Hydrocarbon-degrading marine bacteria relevant in the
nitrogen cycle
Elena GARCÍA-VALDÉS, Zoyla DAVID, Magdalena MULET, Aina CLADERA,
Joan MAYOL and Jorge LALUCAT
Departament de Biologia and IMEDEA (CSIC-Universitat de les Illes Balears). Campus UIB, 07122 Palma
de Mallorca, Spain
([email protected]; [email protected]; [email protected])
ABSTRACT
Hydrocarbon-degrading bacterial strains have been isolated from contaminated sand as the result
of the Prestige oil spill. Those microorganisms able to denitrify have been selected. These strains, together with other
denitrifying degraders, or strains able to grow diazotrophically selected from our culture collection, have been
presumptively identified through the partial 16S rDNA sequence. Strains were physiological characterized as the basis
for conducting laboratory microcosms experiments, to follow the crude oil degradation and changes in the bacterial
community under different environmental conditions.
1. INTRODUCTION
Crude oil spills have a great impact on the bacterial communities in natural ecosystems,
modifying their composition due to their toxic effect against many of them, and stimulating the
growth of hydrocarbon degraders. Bacterial communities are the first to react in front the
presence of contaminants. The growth of degraders is usually limited by the amounts of
nutrients needed for the catabolism of the hydrocarbons and for the synthesis of new biomass.
Oxygen and nitrogen are limiting factors in the biodegradation rates of the contaminants.
Bacteria play key roles not only as degraders, but also in the nitrogen cycling of elements in
natural ecosystems, acting as nitrogen fixers and as denitrifiers, affecting in this way the
biodegradation rates. To gain inside on the impact of the oil spill on the bacterial communities in
the coast, two physiological groups of bacteria able to grow aerobically on components of crude
oil have been studied in our culture collection of microorganisms and in natural samples taken
in the Galicia coast. In this way, models can be designed in the laboratory to assess the role of
each physiological group, and we will be able to compare the communities from contaminated
and non-contaminated areas in the Galicia coast. Groups of aerobically hydrocarbon-degraders
studied were: 1) Bacteria also able to grow anaerobically as denitrifiers. 2) Bacteria able to
grow aerobically and diazotrophically, fixing nitrogen. The isolates were characterised for their
ability to grow on hydrocarbons and taxonomically by the analysis of the 16S rRNA sequences.
2. RESULTS AND DISCUSION
1. Samples. Sand samples were taken in March 2004 in Lariño beach (Galicia, Spain)
highly polluted with crude oil as a consequence of the Prestige oil spill. This part of the coast
was not fertilized previously with nutrients, nor bioaugmentation experiments had been
performed.
2. Isolation of denitrifying bacteria. A selective oligotrophic medium with KNO 3 as terminal
electron acceptor was used for enrichment or direct isolation under anaerobic conditions.
Maltose, starch, glycerol and ethylene-glycol at 0.5% were used as substrate. Incubation
temperatures were 20 and 30oC. Isolates were tested for denitrification by culture on liquid
medium with the same carbon source and Durham tubes to detect the formation of gas from
nitrate.
Five pure cultures with different colonial morphologies were obtained when the sample was
resuspended in Ringer and directly inoculated on plates, without previous enrichment. Four
strains were isolated after enrichment in oligotrophic and copiotrophic selective medium for
denitrifiers. Twenty-two strains were isolated after previous aerobic enrichment in mineral
medium with naphthalene (9) or hexadecane (13) as the only carbon source in oligotrophic
medium.
VERTIMAR-2005
Symposium on Marine Accidental Oil Spills
3. Presumptive identification. Partial 16S rRNA sequences (400-1000 nucleotides) were
determined by standard procedures previously described (Cladera et al., 2004). A BLAST
analysis on nucleotide sequences databases, together with the construction of phyogenetic
trees, affiliated most of the isolates to described species or genera of marine bacteria.
Microbacterium: M oxydans, M. maritypicum
Marinobacter
Nocardioides simplex
Ochrobactrum
Oerskovia turbata
Rhodococcus
Pseudomonas: P. fulva, P. putida, P. pachastrellae
The isolates were classified taxonomically and their capacity of growth on hydrocarbons
(hexadecane and naphthalene) was also tested for each isolate.
4. Selection of denitrifiers and nitrogen-fixers from culture collection. Previously isolated
marine hydrocarbon-degraders of the species Pseudomonas stutzeri able to grow anaerobically
with nitrate were selected for further studies. The collection included naphthalene, phenantrene,
hexadecane and monoaromatic-degraders. Some of them could grow anaerobically with nitrate
on monoaromatics, and others utilized both, aromatics and alcanes aerobically (Song et al.,
2000). Strains B1SMN1 and S1MN1 were isolated from a waste water lagoon, and are able to
grow diazotrophically on naphthalene, and are independent of combined nitrogen sources
(Rosselló et al., 1991). This collection of strains was characterized taxonomically and with
respect their denitrification and nitrogen fixation ability.
Studies on the fate in microcosms experiments of these autochthonous marine bacteria
isolated from the Galicia coast, together with selected culture collection strains, are under way.
REFERENCES
Cladera AM, A Bennasar, M Barceló, J Lalucat, E García-Valdés (2004) Comparative genetic
diversity of Pseudomonas stutzeri genomovars, clonal structure and phylogeny of the
species: J.Bacteriol. Vol. 186 nº16 p. 5239-5248.
B, Norberto J. Palleroni, Max M. Häggblom (2000) Isolation and characterization of diverse
halobenzoate degrading denitrifying bacteria from soils and sediments. Appl. Environ.
Microbiol Vol: 66 nº8 p. 3446-3453.
Rosselló R, E García-Valdés, J Lalucat, J Ursing (1991) Genotypic and phenotypic diversity of
Pseudomonas stutzeri. System. Appl. Microbiol. 14, p.150-157.
Acknowledgements
This work was supported by grant VEM2003-20565/INTER from the CICYT, Spain. We
thank NJ Palleroni for supplying bacterial strains. Researchers of the Instituto de
Investigaciones Marinas (CSIC-Vigo) are acknowledged for helping in deciding the sampling
area.