Escherichia coli translocates formate/formic acid bidirectionally across the cytoplasmic membrane by the FocA/FocB formate channels during fermentation. Depending on the pH and whether formate is supplied exogenously or generated internally, the mechanisms of translocation differ. This study elucidates the role of these channels in dependence on F_OF₁ ATPase activity in stationary phase cells after cultivation by mixed-carbon fermentation at pH 7.5. In cells cultivated with glucose plus glycerol, exogenously added formate increased the N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive (F_OF₁ ATPase-dependent) proton flux in single or double foc mutants. Moreover, exogenously supplied formate also increased the DCCD-sensitive potassium flux, but only in mutants where focB was absent. In the cells grown on glucose, glycerol, and formate, addition of formate in the whole-cell assays increased F_OF₁ ATPase activity by ∼60% compared with cells grown on a mixture of only glucose and glycerol. In a focA mutant cultivated to the stationary phase on glucose, glycerol, and formate, F_OF₁ ATPase activity was double that compared with cells grown on only glucose and glycerol, while in a focA-focB double-null mutant F_OF₁ ATPase activity decreased by ∼50% in formate assays. These data suggest that the cell regulates the mechanism of formate translocation depending on whether formate is generated internally or added exogenously. Thus, F_OF₁-ATPase activity and the FocA/FocB channels together with formate hydrogenlyase activity combine to balance pH and ion gradients during fermentation in stationary phase cells in response to whether formate is generated metabolically or supplied in high concentration from the environment.

Various pretreatment methods for the valorization of sunflower husks (SHs) for H₂ gas generation through fermentation by Escherichia coli were investigated. We analyzed thermal treatment (TT), acid hydrolysis (AH), and alkaline hydrolysis (AlkH) at different substrate concentrations (50 g L⁻¹, 75 g L⁻¹, 100 g L⁻¹, and 150 g L⁻¹) and dilution levels (undiluted, 2× diluted, and 5× diluted). A concentration of 75 g L⁻¹ SH that was acid-hydrolyzed and dissolved twice in the medium yielded optimal microbial growth, reaching 0.3 ± 0.1 g cell dry weight (CDW) L⁻¹ biomass. The highest substrate level enabling effective fermentation was 100 g L⁻¹, producing 0.37 ± 0.13 (g CDW) × L⁻¹ biomass after complete fermentation, while 150 g L⁻¹ exhibited pronounced inhibitory effects. It is worth mentioning that the sole alkaline treatment was not optimal for growth and H₂ production. Co-fermentation with glycerol significantly enhanced both biomass formation (up to 0.42 ± 0.15 (g CDW) × L⁻¹)) and H₂ production. The highest H₂ yield was observed during batch growth at 50 g L⁻¹ SH hydrolysate with 5× dilution, reaching up to 5.7 mmol H₂ (g sugar)⁻¹ with glycerol supplementation. This study introduces a dual-waste valorization strategy that combines agricultural and biodiesel industry residues to enhance clean energy generation. The novelty lies in optimizing pretreatment and co-substrate fermentation conditions to maximize both biohydrogen yield and microbial biomass using E. coli, a widely studied and scalable host.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H₂ and CO₂. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H₂-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H₂-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H₂ production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

In Escherichia coli, the FhlA acts as a transcriptional activator for fdhF , hyc and hyp operons, whose gene products constitute the formate hydrogen lyase (FHL) complexes. These complexes contribute to bioenergetic regulation by consuming intracellular protons during the conversion of formate to hydrogen gas (H ₂ ). The current study elucidates the role of the FHL complex (using fhlA mutant) in the proton transport and energy transduction during the fermentation of glucose, glycerol and formate at pH 5.5. It was shown that F _O F ₁ -ATPase activity and proton flux rate were decreased in fhlA at 20 h and 72 h grown cells, compared to WT indicating the functional interplay between FHL and F _O F ₁ and highlighting its importance in proton transfer at acidic conditions. Increased number of membrane –SH groups and decreased proton conductance (C _M H ⁺ ) value in both WT and fhlA mutant were detected at 72 h, compared to 20 h, suggesting efficient energy transduction at acidic pH when H ₂ was not generated. The value of membrane potential (ΔΨ) remained unchanged in FhlA-lacking cells and was independent of growth time. Thus, bacteria regulate proton motive force (Δp) managing bioenergetic association between proton ATPase activity, FHL function and transmembrane proton gradient (ΔpH) regulation. Overall, these findings demonstrate that the FHL complex plays an essential role in coordinating proton flux, regulation of proton motive force and energy transduction in E. coli under acidic fermentative conditions through functional interplay with the F _O F ₁ -ATPase.