As microbial sulfur metabolism significantly contributes to the formation and cycling of deep-sea sulfur, studying their sulfur metabolism is important for understanding the deep-sea sulfur cycle. However, conventional methods are limited in near real-time studies of bacterial metabolism. Recently, Raman spectroscopy has been widely used in studies on biological metabolism due to its low-cost, rapid, label-free, and nondestructive features, providing us with new approaches to solve the above limitation. Here, we used the confocal Raman quantitative 3D imaging method to nondestructively detect the growth and metabolism of Erythrobacter flavus 21-3 in the long term and near real time, which possessed a pathway mediating the formation of elemental sulfur in the deep sea, but the dynamic process was unknown. In this study, its dynamic sulfur metabolism was visualized and quantitatively assessed in near real time using 3D imaging and related calculations. Based on 3D imaging, the growth and metabolism of microbial colonies growing under both hyperoxic and hypoxic conditions were quantified by volume calculation and ratio analysis. Additionally, unprecedented details of growth and metabolism were uncovered by this method. Due to this successful application, this method is potentially significant for analyzing the in situ biological processes of microorganisms in the future. IMPORTANCE Microorganisms contribute significantly to the formation of deep-sea elemental sulfur, so studies on their growth and dynamic sulfur metabolism are important to understand the deep-sea sulfur cycle. However, near real-time in situ nondestructive metabolic studies of microorganisms remain a great challenge due to the limitations of existing methods. We thus used an imaging-related workflow by confocal Raman microscopy. More detailed descriptions of the sulfur metabolism of E. flavus 21-3 were disclosed, which perfectly complemented previous research results. Therefore, this method is potentially significant for analyzing the in-situ biological processes of microorganisms in the future. To our knowledge, this is the first label-free and nondestructive in situ technique that can provide temporally persistent 3D visualization and quantitative information about bacteria.
Keywords: confocal Raman microscopy; deep-sea bacteria; in situ; near real time; sulfur metabolism.