Beyond traditional pathways: Uncovering ultrasonication's novel routes to enhance hydrogen yields in Clostridium pasteurianum

This study presents a comparative analysis of metabolic end-products and proteomic profiles in Clostridium (C.) pasteurianum during dark fermentation, in the presence and absence of ultrasonication. The study revealed significant changes in the abundance of six proteins associated with dark fermentation pathways, particularly those involved in hydrogen generation pyruvate:ferredoxin oxidoreductase (PFOR) and pyruvate formate lyase (PFL)), glycolysis, butyrate pathway, and adenosine triphosphate (ATP) synthesis. Ultrasonication was efficient at degassing with dissolved hydrogen concentrations reduced from 2.63 ± 0.09 mM to 0.65 ± 0.04 mM. This led to an 11 % increase in produced hydrogen concentration, and a 37 % increase in hydrogen yield. Contrary to expectations, ultrasonication resulted in a deceleration of both glycolytic and hydrogen generation pathways, as evidenced by a decrease in the proteomic expression of key enzymes like pyruvate:ferredoxin oxidoreductase and pyruvate formate lyase. The study also discovered an increased ATP demand and an altered carbon flux through metabolic pathways in C. pasteurianum. This flux initially favoured the acetate pathway due to the sudden ATP demand. The long-term maintenance of this ATP demand required balancing the carbon flux between lactate, acetate, and butyrate pathways, to achieve both a stable redox balance and ATP demand. For the first time, this study revealed opportunities to enhance hydrogen yields using strategies other than enhancing the hydrogen generation pathways, specifically by reducing dissolved hydrogen concentrations.