pubs.acs.org/JAFC Published on Web 10/25/2010 © 2010 American Chemical Society 11680 J. Agric. Food Chem. 2010, 58, 11680–11687 DOI:10.1021/jf102514r Chemical Structures of Corn Stover and Its Residue after Dilute Acid Prehydrolysis and Enzymatic Hydrolysis: Insight into Factors Limiting Enzymatic Hydrolysis J.-D. MAO,* ,† K. M. HOLTMAN, ‡ AND D. FRANQUI-VILLANUEVA ‡ † Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Blvd, Norfolk, Virginia 23529, United States, and ‡ Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, United States Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced solid-state NMR spectral-editing techniques as well as 1 H- 13 C two-dimensional heteronuclear correlation NMR (2D HETCOR) were employed. Our results indicated that dilute acid prehydrolysis selectively removed amorphous carbohydrates, increased aromatic CH/other protonated -CdC- and enriched alkyl CH and CH 2 components. Cinnamic acids were increased, and proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated in RES. 2D HETCOR experiments indicated a close association between lignin and the residual carbohydrates. Ketones/ aldehydes were not detected in the DAP, in contrast to a report in which an appreciable amount of ketones/aldehydes was generated from the acid pretreatment of a purified cellulose in the literature. This suggested that acid pretreatment may modify the structure of purified cellulose more than biomass and that biomass may be a better substrate than model biopolymers and compounds for assessing structural changes that occur with industrial processing. On the basis of NMR and wet chemical analyses, we found the following factors could cause the limitations to the effectiveness of enzymatic hydrolysis: (1) chemical modification of carbohydrates limited the biologically degradable carbohydrates available; (2) cinnamic acids in the residue accumulated; (3) accessibility was potentially limited due to the close association of carbohydrates with lignin; and (4) proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated. KEYWORDS: Corn stover; NMR; bioethanol; biomass; lignin; cellulose INTRODUCTION It is well-known that cellulose in plant materials such as corn stover is encrusted with hemicellulose and lignin in the cell wall. Cellulose conversion is directly related to the removal of hemi- cellulose and lignin, creating a topochemical effect which opens pore space in the cell wall structure and allows for enhanced accessibility to the cellulose ( 1 -5 ). One of the predominating technologies proposed for conversion of this biomass is acid prehydrolysis which has been extensively evaluated by the National Renewable Energy Laboratory (NREL) ( 6 -8 ). Acid prehydrolysis solubilizes a significant portion of hemicellulose and some lignin, lowers the degree of polymerization of the cellulose through endwise hydrolysis and random chain scission, creates new reducing end units, and renders the cellulose easily accessible to enzymatic attack. It has been widely reported that the extent of glucose yield is limited to 70% by dilute acid hydrolysis techniques ( 9 -12 ), although Torget et al. ( 13 ) have reported that an 85% conversion with a shrinking bed percolation reactor was possible. The chem- ical factors that limit this yield could include glucose degrada- tion ( 14 ), glucose reversion reactions ( 9 ), cellulose degradation via parallel parasitic pathways ( 15), modification of cellulose poly- mers or oligomers ( 10 , 11, 15, 16), and repolymerization reactions with lignin intermediates and carbohydrate byproduct ( 17, 18 ). Such studies typically utilize monomers or low-molecular-weight materials which are readily solubilized and hence more easily analyzed. However, in order to enhance the production of ethanol, it is necessary to understand the underlying reasons for the limitations to hydrolysis more clearly by directly studying the insoluble residues after pretreatment and enzymatic hydrolysis. Solid-state NMR spectroscopy is considered as the best choice for the analysis of chemical structures of insoluble organic materials since it is non- destructive and can provide comprehensive structural information. In the past years, we have developed, modified, and adopted many advanced solid-state NMR techniques for the detailed, systematic characterization of complex organic matter ( 19 -21 ). Routine 13 C solid-state NMR spectra consist of broad and heavily overlapped bands in which functional groups cannot be clearly distinguished. *To whom correspondence should be addressed. Phone: 757-683- 6874. Fax: 757-683-4628. E-mail: jmao@odu.edu.