Download STRUCTURE AND BIODEGRADIVE POTENTIAL OF BACTERIAL

STRUCTURE AND BIODEGRADIVE POTENTIAL OF BACTERIAL COMMUNITIES IN OILPOLLUTED COASTAL MARINE SEDIMENTS
Nadezhda Hristova Todorova
ABSTRACT
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAH), which are part of the crude oil and oil derivatives,
are among the most widespread anthropogenic pollutants in the coastal zones. In spite of
their importance, little is known about the organic pollution of the Black Sea coasts, the
largest part of which are localized near the Danube river delta. The Strategic Action Plan for
the Environmental Protection and Rehabilitation of the Black Sea 2009 outlined the
increased risk of oil pollution as a priority problem, especially in case of localized chronic
pollutions with weak, but frequent oil discharges.
The biodegradation of PAH by naturally existing bacterial communities is one of the main
mechanisms for the oil elimination in the environment and, as a basic self-clearance
mechanism of affected ecosystems, is of theoretical and practical importance.
The present study applied the methods of the environmental microbiology in determining the
effect of oil pollution on coastal ecosystems and for the first time provided data for the
composition and function of bacterial communities, naturally developed under described
conditions in the Sozopol harbour. The region is characterized by the long-lasting impact of
minor concentrations of oil products due to the summer recreational activities and
commercial fishing. The thesis contains original results on the biodegradive PAH potential in
existing functional bacterial community in oil-polluted sediments.
AIM OF THE STUDY
Characterizing the effect of chronic oil pollution (using molecular methods) on the structure
and biodegradive potential of bacterial communities from coastal sediments near the town of
Sozopol.
MATERIALS AND METHODS
Microcosm laboratory system:
To study the pollutant impact over bacterial community, two laboratory microcosm systems
were designed: pristine and oil-polluted. Coastal sediment and sea water plus organic matter
were mixed in a glass vessel to initialize the development of the available natural bacteria.
To imitate oil pollution, oil has been repeatedly added to the oil microcosm.
Site and type of sampling:
Natural pristine and PAH polluted sediments and water samples were collected from the
coastal zone near Sozopol (SW Bulgarian Black Sea coast), July 2006. Triple undisturbed
sediment core samples were collected from the pristine zone near camping site “Zlatna ribka”
(SZ4) and from the Sozopol harbour (SZ2) via SCUBA diving.
Molecular methods:
DNA isolation from marine sediment and water, PCR amplification of 16S rRNA genes and
cathabolic xylE2 and PAH-RHD genes, ARDRA, clone libraries construction of 16S rRNA
and cathabolic genes, RFLP screening, sequencing.
Analysis
Comparison between fingerprint restriction profiles (ARDRA and RFLP): Pearson correlation
coefficient, UPGMA clustering method, BioNumerics v.6.
Phylogenetic tree construction: Weighted neighbour-joining algorithm from TREE BUILDER.
1
RESULTS
The impact of the chronic PAH pollution on sediment bacterial communities is assessed
using molecular biological methods for the first time in the region.
Initially, the microcosm systems were used to test the pollutant effect. The natural bacterial
communities from pristine (SZ4) and oil-polluted (SZ2) marine sediments were characterized
and compared on the basis of isolated total community DNA. The presence of pollutant was
confirmed by GC-FID analysis.
The community composition from both sites was assessed at two informative levels by
molecular methods. Data about the resident bacteria were gathered by PCR amplification of
16S rRNA gene, with subsequent ARDRA, clone library construction, sequencing and
phylogenetic analysis of a small number of predominant 16S rRNA gene clones. On the first
informative level, ARDRA analysis did not have enough discriminative power to demonstrate
the effect of the pollutant on the 16S rRNA genes and respectively on the structure of
bacterial communities.
The community members were more precisely assessed at the second level by 16S rRNA
gene clone libraries, where the influence of PAH pollution on bacterial communities lead to
differences in their composition: increase of Proteobacteria (and especially -Proteobacteria)
community members along with lack of Actinobacteria affiliated sequences in the oil-polluted
site.
Biodegradive potential of adapted bacterial community in Sozopol oil-polluted harbour
sediments was demonstrated by the presence of PAH-ring hydroxylating dioxygenase gene
fragments (nahAc). The bioavailability of pollutant and the lack of nutrient limitation were
indicated by the lack of dominant phylotypes in the polluted sediments studied. The
comparison of pristine and oil-polluted sediments revealed a functional bacterial community
with PAH-degradive potential from the chronic oil-polluted sediments.
CONTRIBUTIONS OF THE WORK
The present study was the first to evaluate the effect of chronic oil pollution over coastal
sediments near the town of Sozopol and applied molecular methods to reveal the
composition, variety and biodegradive potential of the natural bacterial communities.
Functional bacterial community was revealed from the Sozopol harbour sediments with PAH
levels above the permissible concentrations. The effect of the chronic oil pollution was
revealed via phylogenetic analysis. Only 16S rRNA gene sequences from the polluted
sediments grouped together with database-deposited environmental samples connected to
oil pollution.
A hypothesis about the connection between the dominance of Proteobacteria (especially Proteobacteria) and the chronic oil pollution of coastal sediments was proposed.
The gene fragment of initial PAH-ring hydroxylating dioxygenase gene (nahAc) was
suggested as a biomarker demonstrating the biodegradive potential of bacterial communities
in marine sediments with chronic PAH pollution.
2