Florence Martin
Bacterial diversity exploration in hydrocarbon polluted soil: Metabolic potential and degrader community evolution revealed by isotope labeling
Published on 13 October 2011
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Thesis presented October 13, 2011
Abstract:
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds produced by incomplete combustion of organic matter. They are a source of environmental pollution, especially associated to oil product exploitation, and represent a threat for living organisms including human beings because of their toxicity. Many bacteria capable of degrading PAHs have been isolated and studied. However, since less than 5% of soil bacteria can be cultivated in the laboratory, bacterial species able to degrade PAHs
in situ have been poorly studied. The first goal of this study was to identify bacteria that degrade PAHs in soil using culture-independent molecular methods. To this end, a strategy known a stable isotope probing has been implemented based on the use of phenanthrene, a three rings PAH, in which the natural isotope of carbon was replaced by
13C. This molecule has been introduced as a tracer in microcosms containing soil from a constructed wetlands collecting contaminated water from highway runoff. Bacteria having incorporated the
13C were then identified by 16S rRNA gene sequence analysis after PCR amplification from labeled genomic DNA extracted from soil. The results show that so far little studied Betaproteobacteria, belonging to the genera
Acidovorax,
Rhodoferax,
Hydrogenophaga and
Thiobacillus, as well as Rhodocyclaceae, were the key players in phenanthrene degradation. Predominance of Betaproteobacteria was established thanks to quantitative PCR measurements. A dynamic analysis of bacterial diversity also showed that the community structure of degraders depended on phenanthrene bioavailability. In addition, the phylogenetic diversity of ring-hydroxylating dioxygenases, enzymes involved in the first step of PAH degradation, has been explored. We detected new sequences, mostly related to dioxygenases from Sphingomonadales and Burkholderiales. For the first time, we were able to associate a catalytic activity for oxidation of PAHs to partial gene sequences amplified from soil DNA, by constructing hybrid enzymes and assaying their activity
The results obtained and the tools implemented in this study may be used to develop methods for the diagnostic and monitoring of pollutant biodegradation in processes such as bioremediation of PAHs contaminated sites.
Keywords:
Bacterial diversity, isotopic labeling, 16S rRNA genes, ring-hydroxylating dioxygenases, phylogenetic and functional analysis, qPCR, proteomics, betaproteobacteria, PAHs, phenanthrene, biodegradation
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