Mangrove bacterial diversity and the impact of oil contamination revealed by pyrosequencing: bacterial proxies for oil pollution

Abstract

Background: Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills.

Methodology/principal findings: A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings.

Conclusions/significance: We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest.

Figure 1. Composition of different phyla based on the classification of partial 16S rRNA sequences of bacteria from microcosm sediment using RDP-Classifier.

Phyla (A); Proteobacteria classes (B), Gammaproteobacteria orders (C) and Alteromonadales genera (D). T0 and T23, without oil contamination in the beginning of the experiment and 23 days after oil contamination, respectively; T23 2% and T23 5%, 23 days after 2% or 5% oil contamination, respectively. Roman numerals distinguish the duplicate samples.

Figure 2. Total Petroleum Hydrocarbons (TPH) concentrations during experiment sampling.

T0, T23 0, T23 2%, T23 5% and T66 2%: T0, without oil contamination; T23 0, 23 days after the beginning of the experiment without oil contamination; T23 2%, 23 days after 2% of oil contamination; T23 5%, 23 days after 2% of oil contamination; T66, 66 days after 2% of oil contamination. I and II indicates the duplicates.

Figure 3. Rarefaction curves of partial sequences of 16S rDNA.

The rarefaction curves from microcosm sediment samples, in duplicates, were calculated by DOTUR₀₀₃. T0, T23 0, T23 2%, T23 5% and T66 2%: curves of 16S rDNA of each sampling. T0, without oil contamination; T23 0, 23 days after the beginning of the experiment without oil contamination; T23 2%, 23 days after 2% of oil contamination; T23 5%, 23 days after 2% of oil contamination; T66, 66 days after 2% of oil contamination. I and II indicates the duplicates.

Figure 4. Venn Diagram evaluating the concentration of oil contamination.

In the center of the figure, the Venn diagram is showing unique and sharing OTUs (97%) in each microcosm sample. The order designations of the sequences related to the unique OTUs were determined using the RDP Classifier tool. (A), Unique sequences of samples without oil contamination; (B), Unique sequences of samples contaminated with oil; (C), Unique sequences of samples with 5% of oil contamination; (D), Unique sequences of samples with 2% of oil contamination. ; (E), Unique sequences of samples with 2% and 5% of oil contamination.

Figure 5. Venn Diagram evaluating the time of oil exposure.

In the center of the figure, the Venn diagram is showing unique and sharing OTUs (97%) in each microcosm sample. The order designations of the sequences related to the unique OTUs were determined using the RDP Classifier tool. (A), Unique sequences of samples without oil contamination; (B), Unique sequences of samples contaminated with oil; (C), Unique sequences of samples 66 days after 5% of oil contamination; (D), Unique sequences of samples 23 after 2% of oil contamination; (E), Unique sequences of samples 23 and 66 days after 2% of oil contamination.