A Hybrid DNA-based Technology: Advancing Environmental Investigations for Bioremediation

Bioremediation relies on the natural ability of microorganisms to transform environmental contaminants by using them as growth substrates and energy sources. For its application, it is essential to determine the biodegradative potential of the native microbial community within the contaminated matrix, assessing the presence of specific metabolic capabilities related to pollutant removal. To this end, biomolecular approaches are the most effective. Molecular biology techniques allow for a deeper understanding of the microbial community structure and its specific metabolic functions. Currently, the most widely used technologies include quantitative PCR (qPCR), which enables the absolute quantification of specific taxonomic and catabolic genes, and Next-Generation Sequencing (NGS) of taxonomic markers, which allows for the taxonomic characterization of bacterial communities. However, a major limitation of gene sequencing is that it only provides information on relative abundance, making it difficult to compare samples and different time points. To overcome this issue, starting from sequencing analysis, an innovative analytical methodology has been developed, combining gene sequencing and qPCR (the hybrid DNA-based technology) to improve the resolution and sensitivity of genetic analysis. This approach enables the simultaneous assessment of both the relative abundance of taxa and the absolute quantification of marker genes in environmental samples within a single analysis. This hybrid technology is based on the addition of internal standard genes (ISGs) at a known concentration into the sample before sequencing. The relationship between the relative and absolute quantification of these ISGs obtained by NGS is then used to calibrate the analysis and determine the concentrations of the target marker genes. This approach could be highly promising, both during characterization and monitoring. During the characterization phase, a deep understanding of the microbial community is essential to study its biodegradation potential, allowing for comprehensive statistical assessments of the site's microbial abundance and diversity. Regarding the monitoring phase, the hybrid methodology enables a comparison between samples collected at the same location but at different times, showing the evolution of the bacterial community and in particular regarding of those able to degrade the target contaminants over the monitoring period. The integration of relative and absolute information simplifies result interpretation, enabling more precise and robust conclusions. This, in turn, allows for a more targeted remediation intervention.