Work Packages of BIOREMEDIAT-OMICS

Work Package 1. Dissemination

Aim: To disseminate of the findings of the projects to the scientific community but also to promote the visibility of the project to the local society and target groups which could exploit or being benefited by the project’s findings.

Brief Description:  The scientific dissemination of the project will be achieved via publication of the findings to high quality peer-reviewed journals and by oral and poster presentations to symposia. A key aspect of the project will be the societal dissemination of the findings to the local society, policy makers and industrial stakeholders which could facilitate the commercial uptake of products and services developed during the project.

Work Package 2. Bacterial proteomic analysis

Aim: To identify key enzymes involved in the detoxification of ortho-phenylphenol by S. haloaromaticamans, diphenylamine by P. monteilii and thiabendazole by Sphingomonas sp. through proteomic analysis

Brief Description:  The pesticide-degrading bacteria available will be cultivated in the presence of the relevant pesticides, intermediate metabolites (e.g. benzoic acid or aniline) and glucose (pesticide-free treatment) and the proteome of the relevant bacteria will be analysed using 2-D proteomic analysis. Upregulated or downregulated proteins in response to pesticide exposure will sequenced via LC-MS analysis enabling the identification of key enzymes involved in the metabolism of the relevant pesticides

Work Package 3. Bacterial Genomic Analysis

Aim: To identify via genomic analysis genes with putative role in the metabolism of pesticides by the bacteria isolated.

Brief Description:    Total DNA from active cultures of the ortho-phenylphenol degrading strain S. haloaromaticamans, of the diphenylamine-degrading strain P. monteilii and of the thiabendazole degrading consortium composed of a Sphingomonas sp. and other proteobacteria will be extracted and subjected to genome sequencing via Illumina Miseq. The genome sequencing obtained will be compared to the genomes of all bacteria genomes already available in public databases, assembled and the genes will be annotated.  Genes will putative role in the metabolism of the studied pesticides will be identified and their role will be verified upon comparison with the proteomic data obtained. Further verification for the function of the spotted genes will be obtained via RT-q-PCR analysis

Work Package 4. Soil metagenomic analysis

Aim: to identify novel pesticide catabolic enzymes from contaminated soils using functional metagenomic analysis

Brief Description:  A soil from a wastewater disposal site contaminated with high levels of pesticides like thiabendazole or ortho-phenylpenol will be utilized as a source for the isolation of novel detoxification genes via functional metagenomics. The degrading microflora of the soil will be stimulated with successive additions of the pesticide in question and total DNA will be extracted, fractionated and cloned into appropriate vectors which will be transformed into selected E. coli strains assayed for pesticide-degrading capacities.  Clones showing increasing degradation activity will be further investigated to identify the genes carried in the insert, their organization and their specific function.

Work Package 5. Isolation and characterization of key catabolic enzymes

Aim: to isolate and preliminary characterize key catabolic enzymes carried by the isolated pesticide-degrading bacteria or spotted via soil functional metagenomics

Brief Description:  Work performed in Work Packages 2, 3 and 4 is expected to identify genes involved in the key detoxification steps of the pesticides ortho-phenylphenol, diphenylamine and thiabendazole. Heterologous overexpression of those genes and subsequent purification will provide key catabolic enzymes in pure form which will be preliminary characterized

Work Package 6. Analytical Verification of Pesticides Metabolic Pathway

Aim: to elucidate and confirm the metabolic pathway of the pesticides via analytical means

Brief Description: The data collected in Work Packages 2 and 3 are expected to provide strong indications for the metabolic pathways of the pesticides studied. Confirmation of the metabolic pathways followed for the degradation of ortho-phenylphenol, diphenylamine and thiabendazole by the isolated bacteria will be given by the development and application of advanced analytical methods based on LC-MS/MS and/or GC/MS.

Work Package 7. On site soil bioremediation

Aim: to explore the bioremediation potential of the pesticide-degrading bacterial cultures

Brief Description: Soils collected from sites used for the year round disposal of the wastewaters produced from fruit-packaging plants in Greece and Cyprus will be inoculated with the pesticide-degrading bacteria available and the dissipation of the relevant pesticides will be determined via analytical means.