Integrating real-time imaging and transcriptomics reveals morphotype-associated differences in biofilm architecture and pyrene removal by Mycolicibacterium parafortuitum.

Polycyclic aromatic hydrocarbons are persistent environmental contaminants whose biodegradation is often constrained by low bioavailability, particularly under static and diffusion-limited conditions. This study investigated the association between biofilm architecture and pyrene removal in two closely related Mycolicibacterium parafortuitum strains exhibiting different colony morphotypes. A rough colony morphotype strain, D3 (D3-R), was isolated from agricultural soil using fluorescence-activated cell sorting in a single-cell co-culture with the previously characterized smooth morphotype strain, PO1 (PO1-S). Pyrene removal was monitored by fluorescence imaging and quantified by high-performance liquid chromatography. Comparative genomic analysis showed that both strains possessed complete and highly conserved pyrene degradation pathways despite nucleotide-level variations and differences in mobile genetic elements. Under static conditions, PO1-S exhibited significantly higher (approximately 1.5-fold) pyrene removal than D3-R. Real-time imaging revealed that PO1-S formed flat, laterally spreading biofilms, whereas D3-R developed dense, vertically structured biofilms. Comparative transcriptomic analysis further revealed differential expression of genes involved in extracellular polymeric substance synthesis, cell envelope remodeling, membrane transport, and regulatory functions associated with the contrasting colony morphotypes. Collectively, these findings suggest that differences in biofilm architecture observed between the two closely related strains may contribute to differences in pyrene accessibility and removal under diffusion-limited conditions beyond pyrene degradation gene content alone. These findings provide new insights into the association between biofilm architecture and pyrene removal under static conditions.