Seokwon Jung

Pseudo-lignin and pretreatment chemistry

The formation of pseudo-lignin by the combination of carbohydrate and lignin degradation products has been proposed to be responsible for the increased Klason lignin content in biomass pretreated under acidic conditions. Direct evidence for the presence of pseudo-lignin has never been presented. The formation of additional lignin-like material may be detrimental to enzymatic hydrolysis due to the non-productive binding of enzymes with lignin. To investigate the chemistry of pseudo-lignin formation, dilute acid pretreatments were performed on delignified hybrid poplar biomass under conditions of varying severity. The results show a progressive increase in the Klason lignin content of the acid pretreated material with increasing pretreatment severity. NMR and FT-IR spectroscopic characterization shows the development of aliphatic, unsaturated and carbonyl carbon functionalities in the samples pretreated at higher severities. Given the very low Klason lignin content of the starting material, acid catalyzed dehydration of carbohydrates is responsible for the formation of pseudo-lignin.

Pseudo-lignin formation and its impact on enzymatic hydrolysis

Pseudo-lignin, which can be broadly defined as aromatic material that yields a positive Klason lignin value and is not derived from native lignin, has been recently reported to form during the dilute acid pretreatment of poplar holocellulose. To investigate the chemistry of pseudo-lignin formation, GPC, FT-IR and 13C NMR were utilized to characterize pseudo-lignin extracted from dilute-acid pretreated α-cellulose and holocellulose. The results showed that pseudo-lignin consisting of carbonyl, carboxylic, aromatic and aliphatic structures was produced from dilute acid pretreated cellulose and hemicellulose. Pseudo-lignin extracted from holocellulose pretreated at different conditions had similar molecular weights (Mn∼1000 g/mol; Mw∼5000 g/mol) and structural features (carbonyl, carboxylic, aromatic and methoxy structures). These characterizations have provided the pseudo-lignin formation mechanisms during pretreatment. The presence and structure of pseudo-lignin is important since pseudo-lignin decreases the enzymatic conversion.

Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo‐lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo‐lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT‐IR, and SEM imaging. It was found that hemicelluloses (xylan) derived‐pseudo‐lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ∼ 1.94) dilute acid pretreatment of a xylan–Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo‐lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions. Biotechnol. Bioeng. 2013; 110: 737–753.

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

Direct analysis of cellulose in poplar stem by matrix-assisted laser desorption/ionization imaging mass spectrometry

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was applied to the analysis of the spatial distribution of cellulose on a cross-section of juvenile poplar (Populus deltoids) stems. Microcrystalline cellulose (MCC) was used to optimize matrix (2,5-dihydroxybenzoic acid) application and instrument parameters for the detection of low hexose oligomers, which originated from cellulose in the solid phase. A section of poplar cellulose isolated from juvenile poplar stem which consisted primarily of glucose (∼95%) and minor components such as xylose and lignin was used for the MALDI-IMS studies. The mass spectrum of poplar cellulose consisted of a series of evenly spaced signals having a difference of 162  m/z units, which was similar to that of MCC in linear and reflectron positive ion modes. MS images of cellulose compounds with sodium ion adducts were generated and illustrated the distribution of cellulose on the surface of the poplar stem.

Nanometrology of delignified Populus using mode synthesizing atomic force microscopy

The study of the spatially resolved physical and compositional properties of materials at the nanoscale is increasingly challenging due to the level of complexity of biological specimens such as those of interest in bioenergy production. Mode synthesizing atomic force microscopy (MSAFM) has emerged as a promising metrology tool for such studies. It is shown that, by tuning the mechanical excitation of the probe-sample system, MSAFM can be used to dynamically investigate the multifaceted complexity of plant cells. The results are argued to be of importance both for the characteristics of the invoked synthesized modes and for accessing new features of the samples. As a specific system to investigate, we present images of Populus, before and after a holopulping treatment, a crucial step in the biomass delignification process.

How chip size impacts steam pretreatment effectiveness for biological conversion of poplar wood into fermentable sugars

BackgroundWoody biomass is highly recalcitrant to enzymatic sugar release and often requires significant size reduction and severe pretreatments to achieve economically viable sugar yields in biological production of sustainable fuels and chemicals. However, because mechanical size reduction of woody biomass can consume significant amounts of energy, it is desirable to minimize size reduction and instead pretreat larger wood chips prior to biological conversion. To date, however, most laboratory research has been performed on materials that are significantly smaller than applicable in a commercial setting. As a result, there is a limited understanding of the effects that larger biomass particle size has on the effectiveness of steam explosion pretreatment and subsequent enzymatic hydrolysis of wood chips.ResultsTo address these concerns, novel downscaled analysis and high throughput pretreatment and hydrolysis (HTPH) were applied to examine whether differences exist in the composition and digestibility within a single pretreated wood chip due to heterogeneous pretreatment across its thickness. Heat transfer modeling, Simons’ stain testing, magnetic resonance imaging (MRI), and scanning electron microscopy (SEM) were applied to probe the effects of pretreatment within and between pretreated wood samples to shed light on potential causes of variation, pointing to enzyme accessibility (i.e., pore size) distribution being a key factor dictating enzyme digestibility in these samples. Application of these techniques demonstrated that the effectiveness of pretreatment of Populus tremuloides can vary substantially over the chip thickness at short pretreatment times, resulting in spatial digestibility effects and overall lower sugar yields in subsequent enzymatic hydrolysis.ConclusionsThese results indicate that rapid decompression pretreatments (e.g., steam explosion) that specifically alter accessibility at lower temperature conditions are well suited for larger wood chips due to the non-uniformity in temperature and digestibility profiles that can result from high temperature and short pretreatment times. Furthermore, this study also demonstrated that wood chips were hydrated primarily through the natural pore structure during pretreatment, suggesting that preserving the natural grain and transport systems in wood during storage and chipping processes could likely promote pretreatment efficacy and uniformity.