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.

缺氧条件下的苯降解研究最早于25年前已有报道，但无氧环境下的苯活化机制至今仍不明晰。该领域的研究进展长期受限于厌氧苯降解富集培养物难以稳定培养的瓶颈。本实验室已成功维持一株产甲烷富集培养物，其中包含脱硫杆菌门(Desulfobacterota) ORM2菌株——这是一种独立于所有已知菌属的苯发酵菌，但与其他已报道或预测的苯降解菌存在亲缘关系。ORM2的倍增周期极长（约30天）且接种后延滞期较长，这极大增加了其研究难度。我们为ORM2开发了特异性荧光原位杂交(Fluorescent In Situ Hybridization, FISH)探针，通过该探针观察到ORM2为长度不等的杆状细胞，且倾向于与其他微生物（尤其是产甲烷菌）聚集共生。显微镜观察与基因组学证据表明，ORM2可能分泌胞外聚合物(Extracellular Polymeric Substances, EPS)，这一过程不仅促进细胞聚集，还可能消耗能量从而延长延滞期。值得注意的是，较高的苯浓度（90~120 mg/L）反而会抑制细胞聚集。本研究首次在产甲烷群落中可视化了脱硫杆菌门ORM2菌株的细胞形态，为解析该菌的空间排布规律以及优化其生长速率的潜在策略提供了重要研究思路。