Lignin fractionation and condensation in aromatic-additive-assisted acidic pretreatment and their influence on lignin's effect on the enzymatic hydrolysis

Factors influencing inhibition of lignin on the enzymatic hydrolysis have not been fully elucidated. This study aims to elucidate the effects of lignin fractionation and condensation on its inhibition on enzymatic hydrolysis in aromatic-additive-assisted acidic pretreatment using 2-naphthol (2 N), 2-naphthol-7-sulfonate (NS), and resorcinol (RS). Through simulation reactions of pretreatment and physiochemical analyses of ethanol-extractable lignins (ELs) and cellulolytic enzyme lignins (CELs) from pretreatment, it was observed that 2 N addition in the acidic pretreatment could suppress lignin condensation. This suppression consequently mitigated inhibition of EL-AP-2 N on the enzymatic hydrolysis of Avicel. Simultaneously, addition of NS in the pretreatment can enhance lignin fractionation by facilitating decomposition of lignin into water and ethanol soluble fractions, thereby mitigating inhibition of CEL-AP-NS on the enzymatic hydrolysis. Meanwhile, with RS in the pretreatment, EL-AP-RS and CEL-AP-RS demonstrated the most pronounced inhibition among ELs and CELs. This inhibition may be attributed to the increased phenolic OH groups with the introduction of RS units. A heatmap analysis revealing relationship between lignin characteristics and its inhibition indicated that ELs exhibit reduced inhibition on enzymatic hydrolysis when lignin condensation was suppressed. Conversely, CELs would show diminished inhibitory effects when lignin particle sizes were smaller and lignin fractionation was stronger.