Experimental activities are implemented during the course and are supported by lectures of international experts in the ambit of a series of specific topics that aims at highlighting the facilitation of phytoremediation promoted by microbes. The following activities will be developed during the course and supported by the theoretical lectures:

Field trips. Two field trips will be organized. During the visits the course participants will be introduced to a series of issues related to the history and the characterization of the site and the sampling, including geological and geochemical characterization of the site, strategies for contaminant containment, choice of the sampling site, sampling and sample transport and storage, safety issues. During the field trips the course participants will discuss biotechnological approaches based on the use of microorganisms for enhancing phytoremediation strategies.

A visit to a polluted site and bioremediation facility. Guided tour of a polluted site on national interest. The students will be introduced to field approaches to be used for pollution mapping and monitoring.

A visit to a molecular ecology laboratory at the JRC EU laboratories. Visit to molecular ecology laboratories at JRC: interactive lectures on DNA microarray, metagenomics in environmental biotechnology and monitoring, Visit to the exposition “Landmark in genomics”, visit to “nanoscience lab”.

Establishment and management of phytoremediation microcosms. Plants of different species will be cultivated in microcosms assembled with soil polluted by polychlorobiphenils (PCB). Each student group will receive one microcosm set, and will proceed in bacterial isolation and total DNA extraction from the rhizosphere compartment.

Phylogenetic diversity of bacterial communities in the rhizosphere of plants growing in contaminated soil. The rhizosphere metagenome will be then subjected to different PCR fingerprintings (DGGE, Denaturing Gradient Gel Electrophoresis & ARISA, Amplified Ribosomal Intergenic Spacer Analysis) both in traditional gel and automated capillary electrophoresis, targeting different regions of the ribosomal operons, the 16S rRNA gene and the 16S-23S rRNA intergenic spacers. The results will be statistically analyzed and the structure of the bacterial communities colonizing different cultivars and plants growing in contaminated soil will be evaluated. The metagenome of the polluted soil bacterial community will be analyzed by extensive barcoding pyrosequencing of the 16S rRNA gene. The pyrosequencing libraries will be analyzed by a specific pipeline during the course allowing to identify the species present in the soil before plant stimulation.

Metagenomics of the rhizosphere microbiome. A rhizosphere metagenomic dataset already available in public databases will be analyzed. Students will learn how to assemble genomes, identify and annotate genes assess functional diversity and metabolic pathways in a community metagenome and describe the phylogenetic relationships.

PCB degradation potential of the rhizosphere microbiome. A cultivation independent approach will be applied on the rhizosphere metagenome. Specific genes known to be involved in PCB degradation pathways will be amplified from the metagenome using universal primers. The catabolic potential of the community will be established by quantitative RT PCR of the genes. The presence of the same genes will be screened by PCR on the genomic DNA of bacterial strains isolated from the rhizosphere, in order to establish the degrading potential of cultivable bacteria.

Isolation of rhizosphere strains. A collection of biphenil degrading bacterial isolates will be established from the rhizosphere of the model plants. Since that during the short course it will be possible only to isolate fast growers, the collection will be enriched by slow growing strains isolated from a set of identical microcosms prepared prior to the course.

Characterization of PGP isolates. The strain collection will be dereplicated by ITS fingerprinting to eliminate duplicate isolates, and the selected strains will be identified by 16S rRNA gene sequencing (the 16S rRNA of each strain will be amplified and prepared for subsequent sequencing, the sequences will be available only after the course and will be sent to the course participants). The isolates will be screened in vitro for phenotypes inherent to i) plant growth promotion and colonization, ii) PCB degradation. Isolates harbouring all the tested phenotypes among those listed below will be selected as candidates for PCB phytoremediation processes. Strains harbouring multiple capacities will be selected.

Plant growth promotion activity. The ability to increase nutrient bioavailability (P, Fe) for plant roots, and to stimulate plant growth (ACC deaminase activity, auxine production) will be evaluated through in vitro tests. In vivo tests on the model plant Arabidopsis thaliana will be used for assessing the capacity of the strains of producing VOCs (volatile organic compounds) that can affect plant development by promoting or inhibiting the growth.

Strain genetic labelling. For assessing the capacity of the strains to colonize plant roots transformation trials will be performed with the isolates to develop mutants that are labelled with fluorescent proteins. Plasmids carrying different types of fluorescent proteins (GfP, DsRed) will be used in electroporation experiments.

Plant colonization ability. Fluorescent mutants will be inoculated on plant roots as single strains or in combination or in mix with the natural soil microbes to study the strain capacity of colonizing the rhizoplane and the root interior.

Degradation potential. The strains will be cultivated in mineral media containing biphenyl as unique carbon source. The growth will indicate the capacity of the isolates of degrading the hydrocarbons. PCB degradation will be tested with resting cell assay

Data analyses and paper writing. The results obtained within the course during the last days of the course will be analysed and discussed in guided brainstorming. Working groups will be organized for the preparation of a manuscript draft. The draft will be completed after the course and the summer rest and submitted to an international scientific journal in the fall 2014.

Student’s scientific presentations. All the students participating to the course will give a short talk (10 minutes) presenting their own research topics.