Lignocellulose is a promising renewable resource for producing platform chemicals, such as acetone, butanol, and ethanol, via ABE fermentation by solventogenic clostridia. This study investigates the effects of common lignocellulose derived inhibitory compounds: ferulic acid, coumaric acid, and furfural on Clostridium beijerinckii. Dual-staining with propidium iodide and CFDA, combined with flow cytometry, was employed to assess physiological variability. The results showed that phenolic acid-induced stress helped maintain a higher proportion of viable cells during the production phase, enhancing solvent yields and reducing sporulation. At 0.4 g/L, ferulic and coumaric acids did not reduce cell viability; however, coumaric acid exposure led to an acid-crash profile. Conversely, a more robust inoculum exposed to both phenolic acids simultaneously exhibited effects similar to ferulic acid alone, including slower viability decline, reduced growth and sporulation, and improved solvent production. Furfural exposure at 1.5 g/L resulted in immediate viability loss in 20% of the population, though the overall decline accompanied by the highest sporulation rate occurred later than in the control. Additionally, furfural transformation was slower, suppressing butyrate production and reducing solvent production by 13%. This study suggests that delaying cell death mechanism may explain the stimulatory effects of inhibitors, advancing lignocellulose use in the future.

• Klíčová slova
• Clostridium beijerinckii, ABE, Butanol, Carboxyfluorescein diacetate, Cytometry, Lignocellulose inhibitors, Viability,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Inhibitors that are released from lignocellulose biomass during its treatment represent one of the major bottlenecks hindering its massive utilization in the biotechnological production of chemicals. This study demonstrates that negative effect of inhibitors can be mitigated by proper feeding strategy. Both, crude undetoxified lignocellulose hydrolysate and complex medium supplemented with corresponding inhibitors were tested in acetone-butanol-ethanol (ABE) fermentation using Clostridium beijerinckii NRRL B-598 as the producer strain. RESULTS: First, it was found that the sensitivity of C. beijerinckii to inhibitors varied with different growth stages, being the most significant during the early acidogenic phase and less pronounced during late acidogenesis and early solventogenesis. Thus, a fed-batch regime with three feeding schemes was tested for toxic hydrolysate (no growth in batch mode was observed). The best results were obtained when the feeding of an otherwise toxic hydrolysate was initiated close to the metabolic switch, resulting in stable and high ABE production. Complete utilization of glucose, and up to 88% of xylose, were obtained. The most abundant inhibitors present in the alkaline wheat straw hydrolysate were ferulic and coumaric acids; both phenolic acids were efficiently detoxified by the intrinsic metabolic activity of clostridia during the early stages of cultivation as well as during the feeding period, thus preventing their accumulation. Finally, the best feeding strategy was verified using a TYA culture medium supplemented with both inhibitors, resulting in 500% increase in butanol titer over control batch cultivation in which inhibitors were added prior to inoculation. CONCLUSION: Properly timed sequential feeding effectively prevented acid-crash and enabled utilization of otherwise toxic substrate. This study unequivocally demonstrates that an appropriate biotechnological process control strategy can fully eliminate the negative effects of lignocellulose-derived inhibitors.

• Klíčová slova
• ABE fermentation, Butanol, Clostridium, Fed batch, Ferulic and coumaric acid, Inhibitors, Lignocellulose hydrolysate, Salinity, Wheat straw,
• Publikační typ
• časopisecké články MeSH