Tuesday, May 3, 2011
Dehydrodimerization (termed dimerization from here on) of arabinoxylan ferulate esters crosslink polysaccharides in grass cell walls. Diferulate cross-links impede enzymatic digestion of cell wall polysaccharides to fermentable sugars. Removing diferulate bridges using pretreatments (e.g., Ammonia Fiber Expansion or AFEX) is crucial to optimize saccharification and bioethanol production efficiency. Identification and quantitative profiling of diferulates released during pretreatments provides a revealing view of pretreatment efficiency, but execution of such efforts faces two major challenges. Numerous isomeric diferulates exist that may be released by pretreatment; their similar properties raise the chance of chromatographic co-elution. Another challenge faced when profiling diferulates arises from the chemistry of competing chemical reactions, including hydrolysis and ammonolysis, that release these groups from cell walls. Here we present an ultra-high-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS-MS) approach for diferulate profiling that does not require chemical derivatization. The combination of chromatographic retention time and collision induced dissociation (CID) distinguishes diferulate isomers, and provides the basis for identification and quantification of diferulates released from treated biomass. Extracts of AFEX treated corn stover served as a source of diferulates including diamide, acid-amide, and diacid forms. These analyses detected 8–8-cyclic- and non-cyclic-, 8–O–4-, 8–5-cyclic- (benzofuran-) and non-cyclic-, and 5–5-diferulates in the extracts. More than 90% of the diferulates were released as diferuloyl amides during AFEX pretreatment by ammonolysis and hydrolysis reactions and only about 5% were released as diferulic acids. This reflects relative contributions of ammonolysis (amide forming) and hydrolysis (acid forming) reactions during AFEX.