ETD

• Cladosporium
• detoxification
• mycoremediation
• PCB
• RNA-Seq

Polychlorinated biphenyls (PCBs) are synthetic organochlorine compounds composed of a biphenyl core substituted with varying numbers and arrangements of chlorine atoms, giving rise to 209 distinct congeners. Due to their chemical stability and lipophilic nature, PCBs are classified as persistent organic pollutants (POPs), which persist in the environment and bioaccumulate along the food chain. As a result, they are widely detected in soils and other environmental matrices and represent a significant source of chronic toxicity. Exposure to PCBs has been associated with severe long-term effects in humans, including immunosuppression, neurotoxicity, reproductive disorders, and carcinogenesis. Despite the seriousness of soil contamination, it remains under-recognized by the public and is often described as a “hidden reality” due to difficulties in detection, monitoring, and long-term assessment.
Bioremediation has emerged as a promising strategy for mitigating environmental pollution, and mycoremediation—the use of fungi for pollutant degradation—has gained increasing attention. Fungi are particularly suited for this purpose because of their tolerance to harsh conditions, broad metabolic versatility, and ability to produce powerful oxidative enzymes. Moreover, the use of autochthonous microbial strains isolated from contaminated environments is considered especially effective. Advances in high-throughput sequencing technologies, particularly RNA sequencing (RNA-Seq), enable in-depth investigations of fungal metabolic responses, even in non-model organisms with limited genomic resources.
The aim of this thesis was to apply bioinformatic approaches to transcriptomic data in order to characterize the molecular response of a PCB-degrading fungus exposed to different PCB congeners. Specifically, RNA-Seq was used to analyze global gene expression changes in Cladosporium europaeum F32, a fungal strain isolated from a historically PCB-contaminated soil in France. This strain was selected due to its superior PCB degradation capability compared to other isolates. The study focused on four PCB congeners (52, 101, 153, and 180) and examined transcriptional responses at two time points (24 h and 96 h).
A dedicated RNA-Seq bioinformatic pipeline was developed and optimized for this non-model organism, including quality control, read alignment, gene annotation, expression quantification, and differential expression analysis. The pipeline was based on established workflows and leveraged a reference genome generated previously using long-read PacBio sequencing. Functional annotation and differential expression analysis were performed to identify biological processes potentially involved in PCB response and detoxification.
Fifty RNA samples were analyzed, encompassing control and PCB-exposed conditions. Degradation assays demonstrated active PCB depletion in cultures containing viable fungal mycelium, with the strongest degradation observed for PCB 52 and a moderate reduction for PCB 180, while control conditions remained stable. RNA-Seq generated approximately 1.43 billion high-quality paired-end reads, with mapping rates generally ranging from 60% to 90%. Multivariate analyses revealed that exposure time was the main driver of transcriptional variation, with PCB-specific effects becoming evident only after 96 h. At this time point, 1,139 differentially expressed genes were identified, with PCB 180 eliciting the strongest transcriptional response. Functional enrichment analysis highlighted genes involved in metabolism, stress response, and detoxification, although no direct upregulation of known PCB-degrading genes was observed. Overall, the integration of biochemical and transcriptomic analyses provides valuable insights into fungal responses to PCBs and supports the potential application of Cladosporium europaeum in bioremediation strategies.