We studied the mechanism of the significant enhancement in the enzymatic saccharification of lignocelluloses at an elevated pH of 5.5-6.0. Four lignin residues with different sulfonic acid contents were isolated from enzymatic hydrolysis of lodgepole pine pretreated by either dilute acid (DA) or sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL). The adsorption isotherms of a commercial Trichoderma reesi cellulase cocktail (CTec2) produced by these lignin residues at 50 °C were measured in the pH range of 4.5-6.0. The zeta potentials of these lignin samples were also measured. We discovered that an elevated pH significantly increased the lignin surface charge (negative), which causes lignin to become more hydrophilic and reduces its coordination affinity to cellulase and, consequently, the nonspecific binding of cellulase. The decreased nonspecific cellulase binding to lignin is also attributed to enhanced electrostatic interactions at elevated pH through the increased negative charges of cellulase enzymes with low pI. The results validate the hypothesis that the increases in enzymatic saccharification efficiencies at elevated pH for different pretreated lignocelluloses are solely the result of decreased nonspecific cellulase binding to lignin. This study contradicts the well-established concept that the optimal pH is 4.8-5.0 for enzymatic hydrolysis using Trichoderma reesi cellulose, which is widely accepted and exclusively practiced in numerous laboratories throughout the world. Because an elevated pH can be easily implemented commercially without capital cost and with minimal operating cost, this study has both scientific importance and practical significance.

BackgroundNonspecific (nonproductive) binding (adsorption) of cellulase by lignin has been identified as a key barrier to reduce cellulase loading for economical sugar and biofuel production from lignocellulosic biomass. Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) is a relatively new process, but demonstrated robust performance for sugar and biofuel production from woody biomass especially softwoods in terms of yields and energy efficiencies. This study demonstrated the role of lignin sulfonation in enhancing enzymatic saccharification of lignocelluloses – lignosulfonate from SPORL can improve enzymatic hydrolysis of lignocelluloses, contrary to the conventional belief that lignin inhibits enzymatic hydrolysis due to nonspecific binding of cellulase.ResultsThe study found that lignosulfonate from SPORL pretreatment and from a commercial source inhibits enzymatic hydrolysis of pure cellulosic substrates at low concentrations due to nonspecific binding of cellulase. Surprisingly, the reduction in enzymatic saccharification efficiency of a lignocellulosic substrate was fully recovered as the concentrations of these two lignosulfonates increased. We hypothesize that lignosulfonate serves as a surfactant to enhance enzymatic hydrolysis at higher concentrations and that this enhancement offsets its inhibitive effect from nonspecific binding of cellulase, when lignosulfonate is applied to lignocellulosic solid substrates. Lignosulfonate can block nonspecific binding of cellulase by bound lignin on the solid substrates, in the same manner as a nonionic surfactant, to significantly enhance enzymatic saccharification. This enhancement is linearly proportional to the amount of lignosulfonate applied which is very important to practical applications. For a SPORL-pretreated lodgepole pine solid, 90% cellulose saccharification was achieved at cellulase loading of 13 FPU/g glucan with the application of its corresponding pretreatment hydrolysate coupled with increasing hydrolysis pH to above 5.5 compared with only 51% for the control run without lignosulfonate at pH 5.0. The pH-induced lignin surface modification at pH 5.5 further reduced nonspecific binding of cellulase by lignosulfonate.ConclusionsThe results reported in this study suggest significant advantages for SPORL-pretreatment in terms of reducing water usage and enzyme dosage, and simplifying process integration, i.e., it should eliminate washing of SPORL solid fraction for direct simultaneous enzymatic saccharification and combined fermentation of enzymatic and pretreatment hydrolysates (SSCombF). Elevated pH 5.5 or higher, rather than the commonly believed optimal and widely practiced pH 4.8-5.0, should be used in conducting enzymatic saccharification of lignocelluloses.

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