Visualization of Syntrophic Benzene-Fermenting Desulfobacterota ORM2 in a Methanogenic Enrichment Culture Using Fluorescence In Situ Hybridization

Benzene degradation under anoxic conditions was first reported more than 25 years ago; however, the activation mechanism in the absence of oxygen remains elusive. Progress has been hindered by the difficulty in cultivating anaerobic benzene-degrading enrichment cultures. Our laboratory has sustained a methanogenic enrichment culture harboring Desulfobacterota ORM2, a benzene fermenter distinct from any known genus but related to other known or predicted benzene degraders. ORM2's slow doubling time (∼30 days) and extended lag phase after inoculation complicate its study. We developed a fluorescent in situ hybridization (FISH) probe for ORM2, revealing rod-shaped cells of variable length that tend to cluster with other organisms, particularly methanogens. Microscopy and genomic evidence suggest that ORM2 may produce extracellular polymeric substances, facilitating cell aggregation and possibly consuming energy that contributes to the lag phase. Interestingly, higher benzene concentrations (90-120 mg/L) appeared to reduce cell aggregation. This study visualized the cells of Desulfobacterota ORM2 within a methanogenic community, offering insights into spatial organization and potential strategies to enhance its growth rate.