Abstract :
[en] Optimization of sporulation in probiotic Bacillus species: development of a molecular tool for industrial applications
Bacillus subtilis and Bacillus licheniformis are widely used as probiotics, biostimulants, and biofertilizers due to their ability to form endospores, structures that confer remarkable resistance and make them highly suitable for diverse industrial applications. However, controlling sporulation remains a major challenge: achieving an efficient and reproducible conversion of vegetative cells into endospores on a large scale is difficult, as this complex process strongly depends on the strain, the medium, and the culture conditions.
The aim of this study was to develop a molecular tool enabling real-time monitoring of sporulation initiation in Bacillus, with the goal of rapidly identifying the culture conditions most favorable for efficient industrial-scale endospore production.
To this end, three promoters of key sporulation-related genes (spo0A, spoIIE, spoIIIAA) were selected based on bioinformatic and bibliographic analyses and subsequently fused to the reporter gene gfp-mut3, encoding a variant of the green fluorescent protein. These constructs were introduced into a shuttle plasmid adapted for bacteria of the genus Bacillus.
Among the tested promoters, only that of spoIIE enabled reliable and early detection of sporulation initiation, both in B. subtilis and B. licheniformis, under flask and bioreactor culture conditions. Flow cytometry (FC) analysis revealed strong heterogeneity within bacterial populations at the early stage of sporulation, highlighting the relevance of a monitoring tool at the single-cell level.
In conclusion, the tool developed in this work provides a solid basis for the rapid screening of optimal culture conditions for industrial endospore production. It helps reduce batch-to-batch variability, increase yields, and improve the quality of final products, with direct benefits for human and animal health as well as for agriculture.
