Label-Free, Real-Time Monitoring of Biomass Processing with Stimulated Raman Scattering Microscopy

Research into alternative energy has experienced dramatic growth in recent years, which was motivated by both the environmental impact of current fossil fuels and the unstable and uncertain sources of oil and natural gas. Under ideal conditions, currently unused plant materials, such as agricultural residues, forestry wastes, and energy crops, can be broken down by a series of chemical, enzymatic, and/or microbiological processes into ethanol or other biofuel sources. Biofuels offer an infinitely renewable source of carbon-neutral fuels that can be produced domestically and can make use of waste products from agricultural activity already taking place. The major challenge to be overcome in the widespread adoption of many biofuels is that biomass is intrinsically recalcitrant, making conversion into usable fuels inefficient. This, in turn, means that substantial energy is required to produce the current generation of biofuels, thus decreasing or eliminating their advantages as alternative sources of fuel. The two major chemical species of interest in the biomass conversion process are lignins and polysaccharides such as cellulose and hemicelluloses. Lignins are partly responsible for biomass recalcitrance, but they may also have value as side products in the biorefineries of the future. Cellulose can be broken down to simple sugars, which can then be fermented to produce ethanol. To address the recalcitrance problem presented by lignins, a thermochemical pretreatment process is necessary in current biomass conversion technology. This process uses oxidizing, acidic, or basic conditions along with elevated pressures and/or temperatures to remove or modify lignins and hemicelluloses, thereby enhancing the accessibility for the cellulase enzymes used in the breakdown of cellulose. 6] To optimize the overall conversion efficiency, a detailed understanding of the hydrolysis kinetics of polysaccharides and lignins is critical. For this reason, analytical tools to study the biomass conversion process are needed. Herein, we demonstrate that stimulated Raman scattering (SRS) microscopy, a new imaging method, can offer new information on the biomass conversion processes. The ideal technique for studying the conversion process in situ should offer chemical specificity without exogenous labels, non-invasiveness, high spatial resolution, and real-time monitoring capability. Current analytical methods, such as gas chromatography–mass spectrometry, electron or scanning-probe microscopy, and fluorescence microscopy, cannot satisfy all of these requirements. Microscopy based on infrared absorption offers chemical specificity, but the spatial resolution is limited by the long infrared wavelengths, and penetration depth into aqueous plant samples is limited. Raman microspectroscopy is widely used because it offers label-free chemical contrast with high resolution and chemical specificity. However, the Raman scattering effect is weak, and long pixel dwell times (on the order of 0.1–1 s) are required for imaging plant materials. This means that real-time imaging is challenging, as even a 256! 256 pixel image would require almost two hours at 0.1 s/pixel. Consequently, the dynamic processes involved in the conversion cannot be followed at high spatiotemporal resolution. Coherent Raman microscopy techniques solve many of these problems and offer label-free chemical imaging with high sensitivity and high spatial resolution. Coherent antiStokes Raman scattering (CARS) microscopy is a technique that has been developed over the past ten years and applied to numerous problems of biological or biomedical relevance. However, CARS microscopy suffers from a nonresonant electronic background that can distort the chemical information of interest, making quantitative image interpretation challenging. Herein, we introduce stimulated Raman scattering (SRS) as a tool to study biomass conversion. SRS [*] B. G. Saar, Prof. X. S. Xie Department of Chemistry and Chemical Biology Harvard University, Cambridge, MA (USA) Fax: (+1)617-496-8709 E-mail: xie@chemistry.harvard.edu Y. Zeng, Y. Liu, M. E. Himmel, S. Ding Biosciences Center, National Renewable Energy Laboratory Golden, CO (USA) and Bioenergy Science Center, Oak Ridge National Laboratory Oak Ridge, TN (USA) Fax: (+1)303-384-7752 E-mail: shi.you.ding@nrel.gov C. W. Freudiger Department of Physics and Department of Chemistry and Chemical Biology Harvard University, Cambridge, MA (USA) [**] We thank G. R. Holtom and M. B. J. Roeffaers for helpful discussions. B.G.S. was supported by the Army Research Office through an NDSEG fellowship. C.W.F. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. This work is also supported by the US Department of Energy: the instrumentation and data analysis is funded under grant DE-FG02-07ER64500, and the BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological & Environmental Research in the DOE Office of Science; the delignification process is funded by the Office of the Biomass Program. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201000900. Communications