Candice E. Halbert

Neutron Reflectometry and QCM-D Study of the Interaction of Cellulases with Films of Amorphous Cellulose

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) of the interaction of a fungal enzyme extract ( T. viride ) and an endoglucanse from A. niger with amorphous cellulose films. The use of amorphous cellulose is motivated by that the fact that several biomass pretreatments currently under investigation disrupt the native crystalline structure of cellulose and increase the amorphous content. NR reveals the profile of water through the film at nanometer resolution and is highly sensitive to interfacial roughness, whereas QCM-D provides changes in mass and film stiffness. NR can be performed using either H(2)O- or D(2)O-based aqueous reservoirs. NR measurement of swelling of a cellulose film in D(2)O and in H(2)O revealed that D/H exchange on the cellulose chains must be taken into account when a D(2)O-based reservoir is used. The results also show that cellulose films swell slightly more in D(2)O than in H(2)O. Regarding enzymatic digestion, at 20 °C in H(2)O buffer the T. viride cocktail rapidly digested the entire film, initially roughening the surface, followed by penetration and activity throughout the bulk of the film. In contrast, over the same time period, the endoglucanase was active mainly at the surface of the film and did not increase the surface roughness.

Structural Characterization of a Model Gram-Negative Bacterial Surface Using Lipopolysaccharides from Rough Strains of Escherichia coli

Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air–liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m–1 and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m–1, distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model.

Interactions of endoglucanases with amorphous cellulose films resolved by neutron reflectometry and quartz crystal microbalance with dissipation monitoring

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.

Depletion at solid/liquid interfaces: Flowing hexadecane on functionalized surfaces

We present a neutron reflectivity study on interfaces in contact with flowing hexadecane, which is known to exhibit surface slip on functionalized solid surfaces. The single crystalline silicon substrates were either chemically cleaned Si(100) or Si(100) coated by octadecyl-trichlorosilane (OTS), which resulted in different interfacial energies. The liquid was sheared in situ and changes in reflectivity profiles were compared to the static case. For the OTS surface, the temperature dependence was also recorded. For both types of interfaces, density depletion of the liquid at the interface was observed. In the case of the bare Si substrate, shear load altered the structure of the depletion layer, whereas for the OTS covered surface no effect of shear was observed. Possible links between the depletion layer and surface slip are discussed. The results show that, in contrast to water, for hexadecane the enhancement of the depletion layer with temperature and interfacial energy reduces the amount of slip. Thus a density depletion cannot be the origin of surface slip in this system.

The SNS Liquids Reflectometer

The SNS Liquids Reflectometer [1], installed as one of the first instruments at the Spallation Neutron Source, has now been functional for more than a year. This instrument is designed to view liquid and solid surfaces in specular, off specular, and near-surface small angle scattering geometries. The guide system supplies 2 Å < λ < 16.5 Å neutrons at vertical incident angles ranging from 0° < αi < 5.5° for free liquid surfaces and up to 45° for solid surfaces. Three bandwidth choppers, synchronized with the spallation source and operating at 15–60 Hz, provide neutrons in bandwidths ranging from 3.5–14 Å at a fixed incident angle onto a sample. The sample stage enables all of the motions necessary for positioning liquid and solid surfaces, while the detector arm directs a position-sensitive detector to view the sample at specular or off specular angles up to 90° and can scan out of the specular plane by up to 30°.

Morphological origin for the stratification of P3HT:PCBM blend film studied by neutron reflectometry

Understanding the origin for the film stratification of electron donor/acceptor blend is crucial for high efficiency organic photovoltaic cell. In this study, P3HT:PCBM blend is deposited onto hydrophilic and hydrophobic substrate to examine the film stratifications. The neutron reflectivity results show that, on the different surfaces, PCBM diffuses toward the two interfacial regions in an identical fashion during thermal annealing. This evidences that the film stratification is not affected by the substrates. Instead, since P3HT remains more amorphous in the interfacial regions and PCBM is miscible with amorphous P3HT, PCBM preferentially diffuses to the interfacial regions, resulting in the stratification.

Towards neutron scattering experiments with sub-millisecond time resolution

Neutron scattering techniques offer several unique opportunities in materials research. However, most neutron scattering experiments suffer from the limited flux available at current facilities. This limitation becomes even more severe if time-resolved or kinetic experiments are performed. A new method has been developed which overcomes these limitations when a reversible process is studied, without any compromise on resolution or beam intensity. It is demonstrated that, by recording in absolute time the neutron detector events linked to an excitation, information can be resolved on sub-millisecond timescales. Specifically, the concept of the method is demonstrated by neutron reflectivity measurements in time-of-flight mode at the Liquids Reflectometer located at the Spallation Neutron Source, Oak Ridge National Laboratory, Tennessee, USA, combined with in situ rheometry. The opportunities and limitations of this new technique are evaluated by investigations of a micellar polymer solution offering excellent scattering contrast combined with high sensitivity to shear.

Time-Resolved High Resolution Neutron Imaging Studies at the ORNL Spallation Neutron Source

Our evaluation of a high-resolution digital imaging detector capable of providing millisecond time resolution and high sensitivity for neutrons is presented. This detector is a modified version of the high-resolution gamma-ray imager developed previously at Radiation Monitoring Devices, Inc. (RMD, Inc.), and consists of an Electron Multiplying Charge Coupled Device (EMCCD) attached to a neutron-sensitive scintillator via a fiberoptic taper. By virtue of its internal gain, the EMCCD permits high speed readout without introducing additional noise, thereby enabling high frame rate operation with an enhanced signal-to-noise ratio (SNR). Detector sensitivity is enhanced through the use of a back-thinned EMCCD, which provides high quantum efficiency over a typical emission range for many neutron-sensitive scintillators. Preliminary evaluations conducted at the Liquids Reflectometer beam port of the Spallation Neutron Source (SNS) at the Oak Ridge National Laboratory (ORNL) demonstrate that this new detector has the sensitivity to detect individual neutrons and the acquisition speed to perform energy-selective imaging with a temporal resolution of milliseconds. While substantial improvements in timing and imaging performance are planned, this prototype detector has already generated the first ever images of the SNS Liquids Reflectometer beam profile and was also used to demonstrate a technique for obtaining Bragg edge transmission imaging using energy-selective neutrons. The preliminary data, along with the detector design, evaluation, and planned developments are discussed in this paper.

Interaction of silica nanoparticles with a flat silica surface through neutron reflectometry.

Neutron reflectometry (NR) was employed to study the interaction of nanosized silica particles with a flat silica surface in aqueous solutions. Unlike other experimental tools that are used to study surface interactions, NR can provide information on the particle density profile in the solution near the interface. Two types of silica particles (25 and 100 nm) were suspended in aqueous solutions of varying ionic strength. Theoretical calculations of the surface interaction potential between a particle and a flat silica surface using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory were compared to the experimental data. The theory predicts that the potential energy is highly dependent on the ionic strength. In high ionic strength solutions, NR reveals a high concentration of particles near the flat silica surface. Under the same conditions, theoretical calculations show an attractive force between a particle and a flat surface. For low ionic strength solutions, the particle concentration near the surface obtained from NR is the same as the bulk concentration, while depletion of particles near the surface is expected because of the repulsion predicted by the DLVO theory.

“Old wine in new wineskins:” Upgrading the liquids reflectometer instrument user control software at the Spallation Neutron Source

The Liquids Reflectometer (LR) Instrument installed at the Spallation Neutron Source (SNS) enables observations of chemical kinetics, solid-state reactions, phase-transitions and chemical reactions in general [1]. The ability of the instrument to complete measurements quickly and therefore process many samples is a key capability inherent in the system design [2]. Alignment and sample environment management are a time consuming and error prone process that has led to the development of automation in the control software operating the instrument. In fact, the original LR user interface, based on the Python scripting language, has been modularized and adapted to become the standard interface on many other instruments. A project to convert the original Python [3] implementation controlling the LR instrument into the modular version standardized at SNS was undertaken in the spring of 2012. The key features of automated sample alignment and robot-driven sample management system enable the instrument to reduce the manual labor required to prepare and execute observations, freeing up precious time for analysis and reporting activity. We present the modular PyDas control system [4], its implementation for the LR, and the lessons learned during the upgrade process.