This study investigates the potential of sugar beet pulp (SBP), a lignocellulosic by-product of sugar production, as a low-cost substrate for biohydrogen and biomass generation using Escherichia coli under dark fermentation conditions. Two strains—BW25113 wild-type and a genetically engineered septuple mutant—were employed. SBP was pretreated via thermochemical hydrolysis, and the effects of substrate concentration, dilution, and glycerol supplementation were evaluated. Hydrogen production was highly dependent on substrate dilution and nutrient balance. The septuple mutant achieved the highest H₂ yield in 30 g L⁻¹ SBP hydrolysate (0.75% sulfuric acid) at 5× dilution with glycerol, reaching 12.06 mmol H₂ (g sugar)⁻¹ and 0.28 mmol H₂ (g waste)⁻¹, while the wild type under the same conditions yielded 3.78 mmol H₂ (g sugar)⁻¹ and 0.25 mmol H₂ (g waste)⁻¹. In contrast, undiluted hydrolysates favored biomass accumulation over H₂ production, with the highest biomass yield (0.3 g CDW L⁻¹) obtained using the septuple mutant in 30 g L⁻¹ SBP hydrolysate without glycerol. These findings highlight the potential of genetically optimized E. coli and optimized hydrolysate conditions to enhance the valorization of agro-industrial waste, supporting future advances in sustainable hydrogen bioeconomy and integrated waste biorefineries.

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.

This study examined the growth of E. coli BW25113 wild type in mixtures of sugar beet pulp (SBP) and sugar beet molasses (SBM) waste at various concentrations. The wastes were separately subjected to acid hydrolysis using 0.75% sulfuric acid, with 30 g L-1 SBP and 100 g L-1 SBM. The pH of the solutions was adjusted to 7.5 using potassium dihydrogen phosphate. Experiments were conducted with SBP/SBM ratios of 50/50, 75/25, and 25/75, in undiluted and diluted mixtures (2, 5, and 10 times), with and without 10 mL L-1 glycerol. pH of the medium, oxidation-reduction potential (ORP), and optical density (OD) were measured during batch growth of E. coli BW25113 wild type. The studies were conducted until the 168th hour of growth, with a negative ORP values observed starting from the 3rd hour of growth. It is worth mentioning that when hydrolysates were mixed without dilution no ORP changes and hydrogen production was observed, most possibly due to high concentration of inhibiting agents. Based on the obtained data, optimal conditions for growth and activity were provided by SBP/SBM mixtures at 50/50 and 25/75 ratios, diluted 2-fold, without glycerol, where the maximum hydrogen yield was at 48th and 168th hours, respectively, making up 6.25 mmol L-1 and 5.38 mmol L-1. In the presence of glycerol, based on ORP values, the maximum hydrogen yield in the SBP/SBM 50/50 ratio, 2-fold diluted mixture, was 5.4 mmol L-1 at 24 hours. In the SBP/SBM 25/75 ratio, 2-fold diluted mixture, and SBP/SBM 75/25 ratio, 10-fold diluted mixture, the hydrogen yield was 5.77 mmol L-1 and 5.27 mmol L-1 at 166 and 24 hours, respectively. To sum up 2-fold diluted mixtures with 50/50 and 25/75 ratios, both with and without glycerol, were optimal for growth and hydrogen production.