3D chemical image using TOF-SIMS revealing the biopolymer component spatial and lateral distributions in biomass.

Many researchers consider biofuels, including bioethanol and biodiesel, as a resource to supplement or replace large portions of future transportation fuel requirements. This shift in research focus is due in part to limitations in fossil resources and recent concerns about the environment. Lignocellulosic biomass (for example, agricultural resides, forestry wastes, and energy crops) has been highlighted as a potential resource for biofuel production. Lignocellulosic biomass is mainly composed of polysaccharides (that is, cellulose and hemicelluloses) and lignin (polyphenolic macromolecules). Cellulose, a major source of fermentable sugar used to produce ethanol, is known to be densely packed and embedded in a lignin–hemicellulose matrix. This intricate layering of lignin, cellulose, and hemicelluloses comprises the microstructure of biomass, and has to date not been fully identified. The structural complexity exhibited in lignocellulose originates from innate structural heterogeneity and has been suggested as a contributing factor in the its ability to resist enzymatic hydrolysis, which is referred to as biomass recalcitrance. Biomass recalcitrance has been cited as the major barrier to large-scale utilization of lignocellulosic biomass for biofuel production. Therefore, the major challenge facing future lignocellulosic biofuel research is reducing the recalcitrance of biomass through biological and chemical manipulation. For example, transgenic alfalfa down-regulated in lignin biosynthesis was shown to release more sugar by enzymatic hydrolysis. Thermochemical pretreatment using oxidizing, acidic, or basic conditions under high temperature and/or pressure results in structural cell wall breakdown along with changes in lignin and/or hemicelluloses, ultimately correlating with higher sugar release upon enzymatic hydrolysis. Analytical methods therefore play an important role in determining and understanding changes that occur in biomass during biological and chemical processes designed to reduce biomass recalcitrance. Typically this is carried out with conventional bulk analysis, such as high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC-MS), NMR spectroscopy, and electron microscopy (EM). However, these techniques average over a large spatial dimension, thus losing critical information about differences in chemical heterogeneity as a function of spatial and lateral position in the cell wall. Therefore, we have investigated chemical imaging techniques, which are wellsuited to understand detailed spatial and lateral changes for major components in biomass. Herein, we introduce the first three-dimensional (3D) analysis of biomass using time-of-flight secondary-ion mass spectrometry (TOF-SIMS) in the specific application of understanding recalcitrance. TOF-SIMS is an emerging technique that provides chemical information directly from the surface of biomass without sample treatment, such as matrix application or radioactive labeling. Mass spectra obtained over the sample surface as a result of secondary-ion emission can be mapped into a 2D molecular image representing the lateral distribution of characteristic species at a submicrometer scale. Extending the usefulness of TOFSIMS, a 3D molecular image can be generated by acquiring multiple 2D images in a stack. This is accomplished by reconstruction, which involves stacking the 2D molecular images layer by layer. Each layer is produced in a dual-beam mode, which uses a ion beam for surface analysis and sputtering beam for surface layer ablation, and is also referred to as 3D microarea analysis. As a result, 3D molecular imaging allows both vertical and lateral distributions of targeted or interesting species, from surface to subsurface layers, to be semiquantitively tracked and understood. The ability to capture 3D data seems even more crucial to understanding bioconversion because the interfacial layer between the biomass and cellulolytic enzyme/microbe has been shown to significantly affect hydrolysis. Herein we employ stress-induced tension wood generated on poplar stems as a model substrate to investigate the application of TOF-SIMS on biomass. Tension wood generally appears on the elongated stem side as a result of mechanical bending. Under such conditions, angiosperms form reaction or tension wood on the elongated stem side in an effort to maintain an upright growth position (Figure 1a). Interestingly, tension wood cells have an additional [*] S. Jung, Dr. M. Foston, Prof. Dr. A. J. Ragauskas School of Chemistry and Biochemistry, BioEnergy Science Center, Georgia Institute of Technology 500 10th Street NW Atlanta GA, 30332 (USA) E-mail: Art.Ragauskas@chemistry.gatech. edu