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A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost. 

Lignin depolymerization to aromatic monomers with high yields and selectivity is essential for the economic feasibility of many lignin-valorization strategies within integrated biorefining processes. Importantly, the quality and properties of the lignin source play an essential role in impacting the conversion chemistry, yet this relationship between lignin properties and lignin susceptibility to depolymerization is not well established. In this study, we quantitatively demonstrate how the detrimental effect of a pretreatment process on the properties of lignins, particularly β-O-4 content, limit high yields of aromatic monomers using three lignin depolymerization approaches: thioacidolysis, hydrogenolysis, and oxidation. Through pH-based fractionation of alkali-solubilized lignin from hybrid poplar, this study demonstrates that the properties of lignin, namely β-O-4 linkages, phenolic hydroxyl groups, molecular weight, and S/G ratios exhibit strong correlations with each other even after pretreatment. Furthermore, the differences in these properties lead to discernible trends in aromatic monomer yields using the three depolymerization techniques. Based on the interdependency of alkali lignin properties and its susceptibility to depolymerization, a model for the prediction of monomer yields was developed and validated for depolymerization by quantitative thioacidolysis. These results highlight the importance of the lignin properties for their suitability for an ether-cleaving depolymerization process, since the theoretical monomer yields grows as a second order function of the β-O-4 content. Therefore, this research encourages and provides a reference tool for future studies to identify new methods for lignin-first biomass pretreatment and lignin valorization that emphasize preservation of lignin qualities, apart from focusing on optimization of reaction conditions and catalyst selection.