Adaptive responses of genome-reduced Bacillus subtilis during enzyme secretion in a breathing vessel culture

The utilisation of microbes for the production of enzymes and pharmaceutical proteins is an important step towards a sustainable future. However, the energy requirement for agitation, aeration and cooling during industrial fermentation processes is substantial. To address this challenge, we have previously developed a 'breathing' polytetrafluoroethylene fermenter vessel that allows effective gas exchange with ambient air and, thus, does not require sparger aeration. The present study aimed to explore the potential application of the breathing vessel for enzyme production by the Gram-positive bacterial cell factory Bacillus subtilis. Here, we compared production of the secreted α-amylase AmyQ by the genome-reduced multiple protease-deficient B. subtilis strain IIG-Bs-27-31 during parallel culturing in breathing vessels and shake flasks. Enzyme yields and the cellular and extracellular proteome compositions were assessed.

We observed comparable growth characteristics and AmyQ yields per liter in both culture systems. However, proteome analyses indicated statistically significant differences (p < 0.05, log₂fold change>|0.5|) in the utilization of carbon sources and stress responses between cells grown in the breathing vessels versus shake flasks. In particular, bacteria in the breathing vessel presented activation of the Sigma B-dependent general stress response, presumably to sustain bacterial growth and viability. In contrast, bacteria grown in shake flask presented activated cell envelope stress pathways and typical sheer stress symptoms.

While the overall AmyQ yields per liter were similar in both fermentation systems, the total enzyme yields in the breathing fermenter were significantly higher due to the 15-fold increase in culture volume. Our findings imply that breathing fermentation vessels are suitable for B. subtilis enzyme production and, upon further scale-up, they may represent sustainable and cost-effective alternatives to traditional fermentation systems for microbial cell factories.