CEBEDEAU • Microsens Project

Project background and objectives

The Microsense project was carried out over a 30-month period and funded by the Walloon Region through the Win4Collective program. It brought together two complementary research partners: CEBEDEAU and the Walloon Agricultural Research Centre (CRA-W).

The main objective of the project was to develop innovative, rapid and field-deployable analytical tools for assessing the environmental quality of water (CEBEDEAU) and soil (CRA-W). Unlike conventional analytical approaches that rely on the direct detection of chemical contaminants, Microsense explored an indirect strategy based on the biological response of microbial communities.

This approach is grounded in the assumption that certain microorganisms and functional genes respond specifically to environmental contamination and can therefore serve as bioindicators (bacterial species) or biomarkers (functional genes), reflecting either current or past pollution events.

Selection of pollutants and experimental design

The study focused on plant protection products (PPPs) commonly used in the Walloon Region and considered relevant in terms of current or future environmental pressure. Following a territorial and environmental relevance screening of several dozen compounds, five PPPs were selected: two fungicides and three herbicides.

Laboratory-scale pilot experiments were conducted to identify specific microbial responses to these compounds. Separate bioreactors were fed with wastewater from a treatment plant and exposed to high concentrations of the selected PPPs over several weeks. Regular sampling campaigns were performed to monitor:

• changes in bacterial community composition, and

• variations in gene presence and abundance as a function of the applied treatments.

Bacterial community analysis – 16S metabarcoding

Potential bacterial bioindicators were identified using metabarcoding, based on sequencing of the 16S rRNA gene, a universal phylogenetic marker in bacteria.

Total DNA extracted from the samples was amplified and sequenced to characterize the entire bacterial community. Comparative analyses between control and PPP-exposed conditions enabled the identification of bacterial taxa whose abundance significantly increased or decreased in response to specific pollutants.

Biomarker identification – Metagenomic approach

In parallel, a shotgun metagenomic approach was applied to identify functional gene biomarkers associated with PPP exposure. The entire genetic content of the samples was sequenced, followed by:

• assembly,

• functional annotation, and

• comparative analysis between treatments.

This strategy allowed the identification of genes specifically detected or strongly enriched under certain contamination conditions, particularly genes involved in cell membrane stress responses and aromatic compound degradation pathways.

Key results

Comprehensive bioinformatic analyses led to the identification of:

Bacterial bioindicators

• Dokdonella immobilis, whose abundance systematically increased in the presence of all tested pollutants.

• Pseudoxanthomonas dokdonensis, showing a significant population increase in response to the herbicide S-metolachlor.

Genetic biomarkers

• Four genes associated with cell membrane stress response:

  • K00021 and K01641, detected in the presence of the fungicides fluopyram and prothioconazole;

  • K04708 and K16436, detected in the presence of the fungicides tebuconazole and prothioconazole.

• One gene involved in benzene ring cleavage:

  • ccmtc_K1062, detected in the presence of the herbicide S-metolachlor and the fungicides tebuconazole and prothioconazole.

These biomarkers proved particularly relevant for the detection of triazole fungicide contamination, including both acute (point-source) and chronic pollution scenarios.

Validation and field applicability

To ensure operational relevance, the identified biomarkers were validated using quantitative PCR (qPCR), enabling precise quantification of target gene copy numbers. Specific primer pairs were designed and validated during a second pilot experiment, which included a limited number of pollutants but an increased number of biological replicates to strengthen result robustness.

For field deployment, this approach can be transposed to LAMP-qPCR technology, enabling the development of rapid, portable and cost-effective detection tools.

For bacterial bioindicators, the project explored the development of aptamer-based rapid tests. Aptamers are short DNA fragments capable of binding specifically to bacterial targets and can be integrated into aptasensors, analogous to immuno-based rapid tests. The selection of species-specific aptamers relies on the Cell-SELEX methodology, which is currently under optimization.

Conclusion and perspectives

The Microsense project demonstrated the relevance of environmental microbiology as an indirect monitoring tool for pesticide pollution. The identified biomarkers provide a robust foundation for the development of innovative analytical solutions for water and soil quality monitoring.

Future perspectives include:

• finalizing a proof-of-concept (POC) kit based on LAMP-qPCR technology for the detection of triazole fungicide contamination,

• further optimization of the Cell-SELEX methodology toward the development of a functional aptasensor-based POC for bacterial bioindicators.