Organization of microbial community and fate of pollutants in the rhizosphere – RHIZORG

Rhizotrons and compartmented microcosms were developed to observe root development, sample the soil without destructuration and put in contact the roots with the contaminated soil during at different times.
From extracted soil DNA and RNA and thanks to the high throughput sequencing of markers genes, we access the structure and diversity of microbial communities. 2D geostatistical modeling was performed on many physicochemical and biological parameters.
Microorganisms involved in biodegradation of hydrocarbons were: 1) isolated in pure culture to study their metabolic capacity and their physiology using FT-Raman microspectroscopy ; 2) identified and characterized at the genomic level through the use of 13C-labeled hydrocarbon.

Geostatistical tools allowed visualization of root gradients with depth, and model the differences created by the different plants in the centimeter scale. Bacterial diversity was influenced by the type of plant, the presence or not of plant rhizosphere gradients, and the depth. These gradients of root biomass, pH and nature of exudates explain the spatial structuration of microbial assemblages but not the heterogeneous distribution of hydrocarbons.
On a 10 days kinetic, biodegradation of hydrocarbons was slowed down in the presence of plant, but over a period of more than 1 month, it was almost identical with or without plant and regardless of the type of plant. Thanks to the use of 13C-labeled hydrocarbon, the bacterial species involved in biodegradation were identified by the sequencing of their metagenome. In addition some of these bacteria have been isolated in pure cultures and physiology in the presence or absence of hydrocarbon and in different more or less complex media was studied.

At the end of the project, all these data will lead to a better understanding of the spatial organization and the temporal evolution of microbial communities and the hydrocarbon biodegradation processes in the rhizosphere. We will know better the bacteria involved in the biodegradation of hydrocarbons in situ, as well as their metabolic capacity and physiology in complex ecosystems. We could then extend our findings to other soils, other types of plants and other pollutants.

- Participation to 7 international conferences (including 4 oral presentations and 6 posters)

- Participation to 4 national conferences (including 1 oral presentation and 4 posters)

- Publications in journals of Rank A:
- Bourceret A, Cébron A, Tisserant E, poupin P, Bauda P, Beguiristain T, Leyval C (2016) The bacterial and fungal diversity of an aged PAH- and heavy metal-contaminated soil is affected by plant cover and edaphic parameters. Microbial Ecology. 71(3), 711-724. DOI: 10.1007/s00248-015-0682-8
- Thomas F, Lorgeoux C, Faure P, Billet D, Cébron A (2015) Isolation and substrate screening of polycyclic aromatic hydrocarbon degrading bacteria from soil with long history of contamination. International Biodeterioration & Biodegradation. DOI: 10.1016/j.ibiod.2015.11.004
- Bourceret A, Leyval C, de Fouquet C, Cébron A (2015) Mapping the centimeter-scale spatial variability of PAHs and microbial populations in the rhizosphere of two plants. PlosONE. 10(11): e0142851.
- Thomas F, Cébron A (2016) Short-term rhizosphere effect on available carbon sources, phenanthrene degradation and active microbiome in an aged-contaminated industrial soil. Frontiers in Microbiology. DOI: 10.3389/fmicb.2016.00092

Rhizoremediation (i.e. soil bioremediation assisted by plants) could be an interesting strategy for long-term management of industrial wasteland soils harbouring high organic pollutant concentrations, among which polycyclic aromatic hydrocarbons (PAH). However, studies on PAH-biodegradation in the plant rhizospheres showed contrasted results, with activation as well as inhibition or even no effect of plants on the fate of pollutants. To better understand the processes, we hypothesized that spatial heterogeneities and temporal fluctuations of the plant-soil system would explain the previously observed contrasted results. A soil is heterogeneous due to its intrinsic characteristics, to the pollution and the microorganisms presenting hotspots and plants creating spatial variations around roots (rhizosphere effect). It’s thus important to start taking into account the spatial heterogeneity to better understand the fate of pollutants in planted soils. In the RHIZORG project, all the experiments will be performed on a well-known aged PAH-contaminated soil largely studied in the GISFI consortium (www.gisfi.fr; Neuves-Maisons coking plant wasteland soil, 54, France). The soil will be spiked with a complex pollutant mixture (organic extract from the same soil) to increase pollutant bioavailability and biodegradation. Due to its ability to grow on highly contaminated soils and because it was previously used in phytoremediation studies, ryegrass will be the reference plant used all along the project. The project will focus on chemical analyses of pollutants (PAH and polar aromatic compounds PACs) in parallel with analyses of microbial community diversity and organization within the rhizosphere. The 3 main objectives of the RHIZORG project are to: 1) Determine the spatial and temporal variations of the microbial community and the fate of pollutant during plant growth and root development at a cm-scale, 2) Identify the pollutant-degraders and their metabolic pathways in rhizospheric and bulk soils, and 3) Observe the µm-scale organization and localization of microorganisms and pollutants in the root vicinity. The RHIZORG project will focus on taxonomic and functional bacterial diversity using complementary tools: culture-dependent method (Biolog® MT plates to be develop for organic pollutant degradation metabolic profiles), molecular tools (DNA extraction, fingerprinting TTGE, qPCR, 16S rDNA amplicon pyrosequencing and metagenomic), and microspectroscopic methods (fluorescence, confocal laser scanning and micro-Raman). To meet our objectives new experimental devices need to be designed. The first scientific task aims to study the spatio-temporal variations in the rhizosphere. To do it an original rhizotron will be designed with independent openings for sampling at different times. A regionalized multiple sampling strategy will be used to estimate and map changes in regionalized variables (root biomass, pollutant concentration, bacterial diversity indexes, abundance of functional genes) using geostatistics. The second scientific task is dedicated to the identification of PAH-degrading bacteria and PAH-degradation pathways. Pot devices will be developed to perform Stable Isotope Probing (SIP) on rhizospheric soil and combine the difficulties of using 13C-labeled PAH compound during plant growth. Active functional PAH-degrading populations in plant rhizosphere and bulk soils will be identified, as well as their metagenome, that will tell about specific metabolic pathways involved. The third scientific task has the objective to investigate statistically the spatial organization of the microorganisms and pollutants in the root vicinity using microscopic observation of undisturbed soil thin sections and aims to extrapolate µm-scale organization to the whole root system. Fluorescent in situ Hybridization (FISH) technique will be adapted to undisturbed soil samples and root system will be described using X-Ray tomography.