Effects of heavy fuel oil on the bacterial community structure of a pristine microbial mat

Abstract

The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by "Bacilli" and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution.

FIG. 1.

CA based on T-RFLP data (16S rRNA genes) obtained from control (white triangles) and polluted (black circles) microcosms. The data analyzed correspond to averages for duplicate samples of HaeIII-digested 16S rRNA genes yielding 5′-end T-RFLP patterns. Each number corresponds to the day of sampling relative to the start of the experiment. Encircled symbols correspond to bacterial communities from control samples and from contaminated samples incubated up to 30 days or more than 364 days (the control cluster). Percentage values represent Bray-Curtis similarity among these samples.

FIG. 2.

CA based on T-RFLP data (reverse-transcribed 16S rRNA) obtained from control (white triangles) and polluted (black circles) microcosms. The data analyzed correspond to averages for duplicate samples of HaeIII-digested reverse-transcribed 16S rRNA yielding 5′-end T-RFLP patterns. Each number corresponds to the day of sampling relative to the start of the experiment. Encircled symbols correspond to metabolically active bacterial communities from a control sample at the beginning of the experiment and from control and contaminated samples at the end of the experiment. Percentage values represent Bray-Curtis similarity among these samples.

FIG. 3.

Relative abundance (%) of phylogenetic groups in 16S rRNA clone libraries from control and contaminated microcosms at the beginning of the experiment (0C and 0P, respectively) and from a contaminated microcosm after 90 days of incubation (90P).

FIG. 4.

Phylotype distribution (%) of reverse-transcribed 16S rRNA sequences found in the clone libraries from the different microcosm samples: control (dotted bars) and polluted (striped bars) microcosms sampled just after heavy-fuel-oil contamination and a polluted microcosm sampled 90 days after contamination (solid bars).

FIG. 5.

Phylogenetic tree based on the analysis of reverse-transcribed 16S rRNA cloned sequences (673 bp aligned) from control and contaminated microcosms sampled at the beginning of the experiment (0C and 0P, respectively) and a contaminated microcosm sampled after 90 days of incubation (90P). Only clones (in bold type) and their closest relative sequences affiliated with Gammaproteobacteria are shown. The numbers in parentheses indicate the percentage of clones (difference, ≤1 bp) in the corresponding microcosm library. The tree was rooted with 16S rRNA gene sequences of the strain Desulfovibrio indonesiensis DSM15121^T. The scale bar corresponds to 0.02 substitutions per nucleotide position. Percentages of 100 bootstrap resamplings that supported the branching orders in each analysis are shown above or near the relevant nodes.