Ashutosh Mittal

Effects of alkaline or liquid-ammonia treatment on crystalline cellulose: changes in crystalline structure and effects on enzymatic digestibility.

BackgroundIn converting biomass to bioethanol, pretreatment is a key step intended to render cellulose more amenable and accessible to cellulase enzymes and thus increase glucose yields. In this study, four cellulose samples with different degrees of polymerization and crystallinity indexes were subjected to aqueous sodium hydroxide and anhydrous liquid ammonia treatments. The effects of the treatments on cellulose crystalline structure were studied, in addition to the effects on the digestibility of the celluloses by a cellulase complex.ResultsFrom X-ray diffractograms and nuclear magnetic resonance spectra, it was revealed that treatment with liquid ammonia produced the cellulose IIII allomorph; however, crystallinity depended on treatment conditions. Treatment at a low temperature (25°C) resulted in a less crystalline product, whereas treatment at elevated temperatures (130°C or 140°C) gave a more crystalline product. Treatment of cellulose I with aqueous sodium hydroxide (16.5 percent by weight) resulted in formation of cellulose II, but also produced a much less crystalline cellulose. The relative digestibilities of the different cellulose allomorphs were tested by exposing the treated and untreated cellulose samples to a commercial enzyme mixture (Genencor-Danisco; GC 220). The digestibility results showed that the starting cellulose I samples were the least digestible (except for corn stover cellulose, which had a high amorphous content). Treatment with sodium hydroxide produced the most digestible cellulose, followed by treatment with liquid ammonia at a low temperature. Factor analysis indicated that initial rates of digestion (up to 24 hours) were most strongly correlated with amorphous content. Correlation of allomorph type with digestibility was weak, but was strongest with cellulose conversion at later times. The cellulose IIII samples produced at higher temperatures had comparable crystallinities to the initial cellulose I samples, but achieved higher levels of cellulose conversion, at longer digestion times.ConclusionsEarlier studies have focused on determining which cellulose allomorph is the most digestible. In this study we have found that the chemical treatments to produce different allomorphs also changed the crystallinity of the cellulose, and this had a significant effect on the digestibility of the substrate. When determining the relative digestibilities of different cellulose allomorphs it is essential to also consider the relative crystallinities of the celluloses being tested.

Glucose reversion reaction kinetics.

The reversion reactions of glucose in mildly acidic aqueous solutions have been studied, and the kinetics of conversion to disaccharides has been modeled. The experiments demonstrate that, at high sugar loadings, up to 12 wt % of the glucose can be converted into reversion products. The reversion products observed are primarily disaccharides; no larger oligosaccharides were observed. Only disaccharides linked to the C1 carbon of one of the glucose residues were observed. The formation of 1,6-linked disaccharides was favored, and alpha-linked disaccharides were formed at higher concentrations than beta-linked disaccharides. This observation can be rationalized on the basis of steric effects. At temperatures >140 degrees C, the disaccharides reach equilibrium with glucose and the reversion reaction competes with dehydration reactions to form 5-hydroxymethylfurfural. As a result, disaccharide formation reaches a maximum at reaction times <10 min and decreases with time. At temperatures <130 degrees C, disaccharide formation reaches a maximum at reaction times >30 min. As expected, disaccharide formation exhibits a second-order dependence upon glucose concentration. Levoglucosan formation is also observed; because it shows a first-order dependence upon glucose concentration, its formation is more significant at low concentrations (10 mg mL(-1)), whereas disaccharide formation dominates at high concentrations (200 mg mL(-1)). Experiments conducted using glucose and its disaccharides were calibrated with readily available standards. The kinetic parameters for hydrolysis of some glucodisaccharides could be compared to published literature values. From these experiments, the kinetics and activation energies for the reversion reactions have been calculated. The rate parameters can be used to model the formation of the disaccharides as a function of reaction time and temperature. A new and detailed picture of the molecular mechanism of these industrially important reversion reactions has been developed.

Agave proves to be a low recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands

BackgroundAgave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants.ResultsHere, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon’s Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass.ConclusionsThis study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave’s low recalcitrance for developments in improving cellulosic energy crops.

Hydration and saccharification of cellulose Iβ, II and III I at increasing dry solids loadings

Crystalline cellulose Iβ (Avicel) was chemically transformed into cellulose II and IIII producing allomorphs with similar crystallinity indices (ATR-IR and XRD derived). Saccharifications by commercial cellulases at arrayed solids loadings showed cellulose IIII was more readily hydrolysable and less susceptible to increased dry solids levels than cellulose Iβ and II. Analysis by dynamic vapor sorption revealed cellulose II has a distinctively higher absorptive capacity than cellulose I and IIII. When equally hydrated (g water/g cellulose), low-field nuclear magnetic resonance (LF-NMR) relaxometry showed that cellulose II, on average, most constrained water while cellulase IIII left the most free water. LF-NMR spin–spin relaxation time distribution profiles representing distinct water pools suggest cellulose IIII had the most restricted pool and changes in water distribution during enzymatic saccharification were most dramatic with respect to cellulose IIII compared to celluloses Iβ and II.

Vibrational sum-frequency-generation (SFG) spectroscopy study of the structural assembly of cellulose microfibrils in reaction woods

The cellulose microfibril assemblies in secondary cell walls of tension wood and compression wood were studied with vibrational sum frequency generation (SFG) spectroscopy. The tension wood contains the gelatinous layer with highly-crystalline and highly-aligned cellulose microfibrils. The SFG spectral features of tension wood changed depending on the azimuth angle between the polarization of the incident IR beam and the preferential alignment axis of the cellulose microfibrils. The SFG spectra of the compression wood did not show any dependence on the azimuth angle, implying that the overall orientation of cellulose microfibrils in compression wood is not highly aligned. Instead, the decrease of cellulose content in compression wood brought about larger separation between cellulose microfibrils, which was manifested as changes in CH2/OH intensity ratio in SFG spectra. These results implied that SFG spectral features are sensitive to cellulose microfibril alignments and inter-fibrillar separations.

Investigation of the role of lignin in biphasic xylan hydrolysis during dilute acid and organosolv pretreatment of corn stover

One of the key objectives of biomass pretreatment is to maximize the xylose yield. However, the kinetics of xylan hydrolysis appear to be governed by two parallel first-order reactions with one reaction much faster than the other, thereby limiting both the rate and extent of xylan hydrolysis. Here, we investigate the influence of lignin on xylan hydrolysis kinetics during dilute acid pretreatment of corn stover rind (CSR) by modifying either the substrate or the pretreatment conditions. Dilute acid pretreatment was conducted to test the hypothesis that association of a fraction of the xylan with lignin causes this fraction to hydrolyze at a slower rate resulting in biphasic kinetics. In addition, CSR was pretreated under organosolv (OS) conditions, where xylan and lignin were solubilized simultaneously, to decouple the hydrolysis of xylan from the lignin redistribution process that occurs during dilute acid pretreatment. Dilute acid pretreatment of CSR delignified under mild conditions still exhibited biphasic kinetics, although the fraction of slow hydrolyzing xylan decreased and the rate of fast hydrolyzing xylan increased by 60% resulting in achieving more than 95% total xylose yield. Pretreatment of CSR under OS conditions also appeared to exhibit biphasic xylan hydrolysis kinetics. Unexpectedly, the solubilization of xylan and lignin appeared to occur at similar rates. The increases in the rate and fraction of fast hydrolyzing xylan observed by removing the majority of the lignin support the hypothesis that the slow hydrolyzing xylan is a result of its association with lignin. To further investigate the role of lignin in xylan hydrolysis, the raw and pretreated CSR samples were labeled with a monoclonal antibody (containing a fluorescent dye) that binds xylan specifically so that the location of xylan in the cell wall could be imaged by confocal laser scanning microscopy (CLM). CLM of the pretreated delignified samples showed a similar, intense signal pattern as exhibited by the raw and pretreated control, indicating that the majority of the remaining xylan was located at both cytosolic and middle lamellar cell wall edges. OS pretreated CSR did, however, show a diminution in signal intensity at cell wall edges compared to CSR pretreated under standard dilute acid conditions.

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Here, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.

In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

BackgroundPlant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.ResultsRaman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.ConclusionWe demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

Parameter determination and validation for a mechanistic model of the enzymatic saccharification of cellulose-Iβ

Cost‐effective production of fuels and chemicals from lignocellulosic biomass often involves enzymatic saccharification, which has been the subject of intense research and development. Recently, a mechanistic model for the enzymatic saccharification of cellulose has been developed that accounts for distribution of cellulose chain lengths, the accessibility of insoluble cellulose to enzymes, and the distinct modes of action of the component cellulases [Griggs et al. (2012) Biotechnol. Bioeng., 109(3):665–675; Griggs et al. (2012) Biotechnol. Bioeng., 109(3):676–685]. However, determining appropriate values for the adsorption, inhibition, and rate parameters required further experimental investigation. In this work, we performed several sets of experiments to aid in parameter estimation and to quantitatively validate the model. Cellulosic materials differing in degrees of polymerization and crystallinity (α‐cellulose‐Iβ and highly crystalline cellulose‐Iβ) were digested by component enzymes (EGI/CBHI/ βG ) and by mixtures of these enzymes. Based on information from the literature and the results from these experiments, a single set of model parameters was determined, and the model simulation results using this set of parameters were compared with the experimental data of total glucan conversion, chain‐length distribution, and crystallinity. Model simulations show significant agreement with the experimentally derived glucan conversion and chain‐length distribution curves and provide interesting insights into multiple complex and interacting physico‐chemical phenomena involved in enzymatic hydrolysis, including enzyme synergism, substrate accessibility, cellulose chain length distribution and crystallinity, and inhibition of cellulases by soluble sugars.

Towards an Understanding of Enhanced Biomass Digestibility by In Planta Expression of a Family 5 Glycoside Hydrolase

In planta expression of a thermophilic endoglucanase (AcCel5A) reduces recalcitrance by creating voids and other irregularities in cell walls of Arabidopsis thaliana that increase enzyme accessibility without negative impacts on plant growth or cell wall composition. Our results suggest that cellulose β-1–4 linkages can be cut sparingly in the assembling wall and that these minimal changes, made at the proper time, have an impact on plant cell wall recalcitrance without negative effects on overall plant development.