• bioremediation
• HCH
• Hexachlorocyclohexane
• metabarcoding
• metagenomic
• Metatranscriptomics
• microbiome
• soil

The aim of this thesis is the isolation and characterization of microbiomes competent for the depletion of hexachlorocyclohexane (HCH). HCH is an organochlorinated pesticide worldwide used from the twentieth century for almost 50 years until the banish in 2004 thanks to the Stockholm convention. Despite the documented significance of bioaugmentation in mitigating environmental contamination, current literature remains limited, with few genera being characterized, for HCH degradation.
This lack of information with reference to the diversity in annotated HCH microbial degraders highlights a critical knowledge gap that this research intends to bridge. By analyzing microbial communities across ten distinct soil samples deriving from an historically contaminated site in Italy, the study seeks to expand the repertoire of characterized degraders beyond the already annotated Sphingobium sp.2 with a particular interest in microbiomes. Moreover, in the context of bioaugmentation it is critical to establish efficient molecular approaches to identify and evaluate the microbial taxa involved.
This thesis aims to address these challenges by leveraging metagenome (MG), metatrascriptome (MT) and metabarcoding as predictive tools for monitoring bioaugmentation efficacy in HCH contaminated environments. These advanced molecular techniques might enable the comprehensive profiling of microbial communities, facilitating the identification of key taxonomic markers associated with HCH degradation.
Bioaugmentation introduces specialized microorganisms to accelerate degradation, enhancing the genetic potential of the matrix for remediation. These microorganisms can be allochthonous (from different environments) or indigenous (native to the treated matrix), with indigenous strains often showing better adaptability and survival. Research has highlighted the advantages of using multi-species microbial consortia over single isolates.
Consortia increase the genetic diversity and functional capacity of a matrix, enabling efficient pollutant degradation through complementary metabolic pathways. Synergistic interactions, such as cross-feeding and metabolic division of labor (DOL), enhance degradation. DOL divides complex metabolic processes among community members, reducing the metabolic burden on individual populations and improving overall productivity. Effective degradation of recalcitrant pollutants, such as HCH, depends on the molecule's availability and metabolic intermediates being essential for community survival.
Co-metabolism, where a secondary carbon source enhances the degradation of recalcitrant molecules, might play a critical role. The presence of alternative carbon sources might stimulate microbial growth and enzyme expression, enabling the degradation of contaminants even at low concentrations. This strategy might provide a viable pathway for bioremediation by overcoming energy limitations associated with recalcitrant pollutants. The present work will investigate the co-metabolic strategy in terms of shaping the composition of the degrading microbiomes.
To approach all the objectives described, this research will design and implement a bioinformatics pipeline to process metabarcoding data, facilitating the efficient identification of taxonomic markers linked to HCH degradation activity. This pipeline, which will be made publicly available on GitHub, fulfills a dual purpose: it aids in identifying relevant markers and provides an industrially relevant tool for assessing bioaugmentation performance even in the absence of specific taxonomic markers providing an instrument to identify and quantify the bioaugmented inocula.
The expected outcomes of this thesis include the identification of novel microbial taxa and associated taxonomic markers involved in HCH degradation, as well as the development of a scalable and efficient metabarcoding pipeline for routine use in environmental microbiology and industrial applications. The findings will contribute to a deeper understanding of the microbial ecology underlying HCH degradation, advancing the scientific knowledge of bioaugmentation strategies and their practical implementation in pollutant remediation.
By addressing the limitations in current annotations and proposing a systematic framework for marker identification and bioaugmentation monitoring, this research aligns with the growing need for data-driven approaches in environmental biotechnology. Moreover, the functional characterization of a finely performing microbiome by the MT approach will be adopted to tentatively shed light to the functional characterization of HCH degrading microbiomes and the effects of conservation and mass production on the metagenomic structure.
The scope of this approach is consistent with providing essential information for the implementation of the bioaugmentation protocol that is associated to the need to preserve selected degrading microbiomes for distribution to the scientific community and stakeholders, a lack of information in this direction is actually evident, and to be able to massively grow the selected microbiomes for massive inoculation.