Characterization of sporulation dynamics of Pseudoclostridium thermosuccinogenes using flow cytometry.

Single-cell analysis of microbial population heterogeneity is a fast growing research area in microbiology due to its potential to identify and quantify the impact of subpopulations on microbial performance in, for example, industrial biotechnology, environmental biology, and pathogenesis. Although several tools have been developed, determination of population heterogenity in anaerobic bacteria, especially spore-forming clostridia species has been amply studied. In this study we applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer Pseudoclostridium thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we differentiated and enriched all sporulation-related morphologies of P. thermosuccinogenes. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined culture heterogeneity of P. thermosuccinogenes by discriminating between mature spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study sporulation dynamics and its impact on fermentation performance and product formation by P. thermosuccinogenes.