Visualization of syntrophic benzene-fermentingDesulfobacterotaORM2 in a methanogenic enrichment culture using fluorescencein situhybridization

Abstract

AbstractAlthough benzene degradation under strictly anoxic conditions was first reported over 25 years ago, the mechanism for benzene activation in the absence of oxygen is still elusive. A major limitation has been the difficulty to grow anaerobic benzene-degrading enrichment cultures. Our laboratory has maintained a methanogenic enrichment culture for decades, harboring a benzene fermenter referred to asDesulfobacterotaORM2. Recent genomic analyses indicate that ORM2 is not affiliated with any characterized genus, but it is phylogenetically similar to several other known and predicted benzene degraders.DesulfobacterotaORM2 has a doubling time of approximately 30 days and often enters a long lag or decay phase after inoculation into sterile pre-reduced anaerobic medium. A specific fluorescentin situhybridization (FISH) probe was used to observeDesulfobacterotaORM2 cells during this decay phase, revealing a rod-shaped cell of variable length with a tendency to associate with other cells, particularly methanogens. Microscopic and genomic analyses indicate thatDesulfobacterotaORM2 may produce extracellular polymeric substances (EPS) that likely contribute to cell aggregation. The production of EPS may consume a significant amount of energy, perhaps contributing to the lag time before onset of growth ofDesulfobacterotaORM2 post-inoculation. We observed little cell aggregation in a culture amended with very high concentrations of benzene (90-120 mg/L). This study visualized the cells of a novel clade within theDesulfobacterotafor the first time, enabling monitoring of spatial organization within a methanogenic consortium and provides hints to improve the growth rate of ORM2.ImportanceA specific FISH probe was designed for the poorly characterized benzene fermenterDesulfobacterotaORM2. This probe was used to monitor changes in spatial organization in a methanogenic benzene-degrading enrichment culture. ORM2 cells were often found in cell aggregates, revealing a possible reason for the long lag phases observed after inoculation.