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Thermochemical pretreatment of cellulosic biomass improves cell wall enzymatic digestibility, while simultaneously releasing substantial amounts of soluble oligosaccharides. Profiling of oligosaccharides released during pretreatment yield information essential for choosing glycosyl hydrolases necessary for cost-effective conversion of cellulosic biomass to desired biofuel/biochemical end-products. In this report we present a methodology for profiling of soluble neutral oligosaccharides released from ammonia fiber expansion (AFEXTM)-pretreated corn stover. Our methodology employs solid phase extraction (SPE) enrichment of oligosaccharides based on porous graphitized carbon (PGC), followed by high performance liquid chromatography (HPLC) separation using a polymeric amine based column (Prevail Carbohydrate ES) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in both positive and negative modes. For structural elucidation on the chromatographic time scale, nonselective multiplexed collision-induced dissociation was performed for quasi-simultaneous acquisition of accurate molecular and fragment masses of neutral oligosaccharids in a single analysis. These analyses directly revealed presence of glucans up to degree of polymerization (DP) 22 without side-chain modifications. Additionally, arabinoxylans with DP up to 6 were detected in the pretreated biomass samples (post-enzymatic digestion). All linkages between sugar units in glucans and arabinoxylans were identified to be p-1-4 linkages based on cross-ring fragment masses. Comprehensive profiling of soluble oligosaccharides also demonstrated that arabinoxylan acetylation was reduced by greater than 85% post-AFEXTM treatment.

Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of [M + H]+ ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX���)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals.

Development of environmentally sustainable and economically viable technologies for plant cell wall deconstruction to fermentable sugars has been impeded due to native plant cell wall recalcitrance to thermochemical and biological based processing. Lower severity alkaline-based pretreatment processes like Ammonia Fiber Expansion (AFEX™) can overcome several limitations of traditional pretreatment approaches (e.g., acidic pretreatments) to producing cellulosic biofuels and biochemicals. Here, we give an overview of chemical reactions taking place during alkaline pretreatments including reactions between ammonia and polysaccharides/lignin (e.g., ammonolysis, hydrolysis and Maillard-type reactions). AFEX™ based pretreatments enhance enzymatic digestibility and fermentability of lignocellulosic biomass through various chemical and ultra-structural modifications within the cell wall. An improved mechanistic understanding of the AFEX™ process has led to the development of novel alkaline pretreatments that are briefly discussed in this review.

Accurate quantification of reaction products formed during thermochemical pretreatment of lignocellulosic biomass would lead to a better understanding of plant cell wall deconstruction for production of cellulosic biofuels and biochemicals. However, quantification of some process byproducts, most notably acetamide, acetic acid and furfural, present several analytical challenges using conventional liquid chromatography methods. Therefore, we have developed a high-throughput gas chromatography based mass spectrometric (GC-MS) method in order to quantify relevant compounds without requiring time-consuming sample derivatization prior to analysis. Solvent extracts of untreated, ammonia fiber expansion (AFEX) treated and dilute-acid treated corn stover were analyzed by this method. Biomass samples were extracted with acetone using an automated solvent extractor, serially diluted and directly analyzed using the proposed GC-MS method. Acetone was the only solvent amongst water, methanol and acetonitrile that did not contain detectable background levels of the target compounds or facilitate a buildup of plant-derived residues in the GC injector, which decreased analytical reproducibility. Quantitative results were based on the method of standard addition and external standard calibration curves.