Biohydrogen production with a degenerated strain of Clostridium acetobutylicum ATCC824 from Eichhornia crassipes biomass

Degenerate strains of Clostridium acetobutylicum lack the ability to produce solvents and sporulate and remain in a permanent acidogenic state, allowing continuous hydrogen and organic acid production through anaerobic fermentation. Eichhornia crassipes, an invasive aquatic plant, emerges as a promising source of fermentable sugars for hydrogen production via anaerobic fermentation. In this study, a degenerated strain of Clostridium acetobutylicum was isolated and subsequently cultivated in the presence of a hydrolysate solution obtained from the alkaline pre-treatment and enzymatic hydrolysis of Eichhornia crassipes. The hydrolysate was mixed with a defined medium and served the dual purpose of providing essential nutrients and mitigating inhibitors, eliminating the need for an additional detoxification step. A pure defined culture medium served as a control. The extraction methods employed led to the release of low concentrations of inhibitors, reaching 0.1 g/L of furfural and 0.18 g/L of HMF. Kinetic characterization revealed that in the presence of Eichhornia crassipes hydrolysate, the degenerate strain exhibited lower specific growth rates ranging from 0.114 to 0.156 h⁻¹, compared with the control medium which ranged from 0.131 to 0.179 h⁻¹. This was accompanied by lower yields, ranging from 0.115 to 0.167 g_DCW/g in the presence of hydrolysate versus 0.178 to 0.190 g_DCW/g in the control medium, and diminished butyric acid production of 1.318 to 2.932 g/L in the presence of hydrolysate versus 1.749 to 3.471 g/L in control cultures. Despite reduced growth, high biohydrogen volumetric productivity was achieved, reaching 7.3 L/L·d, along with a significant yield of 2.642 mol of hydrogen per mole of glucose consumed. This represents 66.05% of the maximum stoichiometric yield calculated when acetic acid is the sole byproduct. Apparently, the presence of low concentrations of furfural and HMF released during the pre-treatment of Eichhornia crassipes not only negatively affects growth capacity but also diminishes butyric acid production, favoring biohydrogen production.