Constance A. Schall

Mitigation of Cellulose Recalcitrance to Enzymatic Hydrolysis by Ionic Liquid Pretreatment

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the “overall hydrolysis rates” with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the “primary hydrolysis step” (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster “secondary hydrolysis” (conversion of soluble oligomers to glucose).

Saccharification of ionic liquid pretreated biomass with commercial enzyme mixtures

The performance of Spezyme CP, a commercial cellulase system, and Primafast Luna CL, a textile bio-finishing enzyme, on hydrolysis of substrates following ionic liquid pretreatment was evaluated. Ionic liquid pretreatment of lignocellulosic biomass produces amorphous cellulose with little residual crystallinity and enhances its saccharification. The high crystallinity of native cellulose poses an impediment to enzyme hydrolysis of the cellulosic portion of biomass. Target substrates included poplar, switchgrass and Avicel, a highly crystalline cellulose substrate. Spezyme was found to hydrolyze glucan and xylan components completely in 24h with modest enzyme loadings. Primafast was found to principally hydrolyze glucan components of biomass. Xylanases added to the Primafast enzyme mixture appear to act synergistically to improve glucan hydrolysis with the poplar substrate.

Ionic-Liquid Induced Changes in Cellulose Structure Associated with Enhanced Biomass Hydrolysis

The effects of varying ionic liquid pretreatment parameters on various sources of lignocellulosic biomass have been studied using X-ray powder diffraction, X-ray fiber diffraction, and compositional analysis. Comparative enzymatic hydrolysis and sugar analysis were used to relate the observed changes in cellulose structure to biomass digestibility. In this study, the factor most clearly associated with enhanced biomass hydrolysis is the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase.

Reversible swelling of the cell wall of poplar biomass by ionic liquid at room temperature

Time-resolved autofluorescence, Raman microspectroscopy, and scanning microprobe X-ray diffraction were combined in order to characterize lignocellulosic biomass from poplar trees and how it changes during treatment with the ionic liquid 1-n-ethyl-3-methylimidazolium acetate (EMIMAC) at room temperature. The EMIMAC penetrates the cell wall from the lumen, swelling the cell wall by about a factor of two towards the empty lumen. However, the middle lamella remains unchanged, preventing the cell wall from swelling outwards. During this swelling, most of the cellulose microfibrils are solubilized but chain migration is restricted and a small percentage of microfibrils persist. When the EMIMAC is expelled, the cellulose recrystallizes as microfibrils of cellulose I. There is little change in the relative chemical composition of the cell wall after treatment. The action of EMIMAC on the poplar cell wall at room temperature would therefore appear to be a reversible swelling and a reversible decrystallization of the cell wall.

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass.

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with largely amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH), an endoxylanase (EX) and an acetylxylan esterase (AXE). β-Xylosidase (βX) from Selenomonas ruminantium and a commercial β-glucosidase (βG) from Novozyme 188 were admixed with the A. nidulans enzymes. Statistical analysis indicates that βG and βX activities are significant for both glucose and xylose yields for the two substrates. EG is a significant factor for glucan hydrolysis while EX is significant for xylan hydrolysis of the substrates. The CBH, which has activity on crystalline cellulose and negligible activity on amorphous cellulose, was not a significant factor in glucan hydrolysis. EX is significant in glucan hydrolysis for poplar. The addition of AXE significantly improves xylan hydrolysis for poplar but not switchgrass.

Application of a heat transfer method to determine wax deposition in a hydrocarbon binary mixture

A heat transfer method was used to calculate wax thickness formed from a flowing binary system of hydrocarbons, nonadecylcylohexane and octane. Wax thickness increased steadily with time reaching an asymptotic value. The results obtained with the heat transfer method were in good agreement with the results obtained from gravimetric data. The heat transfer method seems to be a reliable method of calculating wax thickness provided the film heat transfer coefficients in the heat transfer equation can be calculated accurately.

Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals

In experiments conducted at the Bio-CARS beamline 14-BM-C (APS, Argonne National Laboratory, USA), Streptomyces rubiginosus D-xylose isomerase (EC 5.3.1.5) crystals were used to test the effect of cryogen temperature on radiation damage. Crystals cooled using a helium cryostat at an 8 K set temperature consistently showed less decay in the signal-to-noise ratio, I/sigma(I), and in average intensity, I, compared with those cooled with a nitrogen cryostat set to 100 K. Multiple crystals grown using ammonium sulfate as precipitant were used at each cryostat set temperature and comparisons were made for crystals of similar size and diffraction resolution. Maximum resolution for the crystals was 1.1-1.3 A, with He at <20 K extending the lifetime of the high-resolution data by >25% compared with crystals cooled with N(2) at 100 K.

New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.

Cycloalkane solubility determination through differential scanning calorimetry

Abstract Solid–liquid equilibrium data for hydrocarbons are necessary in modeling of paraffin deposition in crude oil production. A multi-cell differential scanning calorimeter (DSC) has been used to measure solubility of pentadecylcyclohexane and nonadecyclohexane in pentane, octane and toluene. The equilibrium solubility temperature of solutions of known composition was determined by a simple linear extrapolation of the characteristic solubility temperature to an infinitely slow heating rate. The thermodynamic properties necessary for solubility prediction (enthalpy of fusion and melting point temperature) were obtained for the pure solutes.

Solvent migration in a paraffin deposit

Temporal changes in solvent content of deposits formed on a tube wall were measured for a binary system of hydrocarbons, nonadecylcylohexane and octane. Solvent content in the deposited nonadecylcyclohexane decreased over time, consistent with the phenomena of migration of included solvent in a temperature gradient. An understanding of inclusion migration in deposits can provide a framework for modeling the aging of paraffin deposits in crude oil pipelines.