Volker S. Urban

Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass

The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to submicrometer length scales resulting from the industrially relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of R(g) ∼ 135 A lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 A, and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the breakdown process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.

Common processes drive the thermochemical pretreatment of lignocellulosic biomass

Lignocellulosic biomass, a potentially important renewable organic source of energy and chemical feedstock, resists degradation to glucose in industrial hydrolysis processes and thus requires expensive thermochemical pretreatments. Understanding the mechanism of biomass breakdown during these pretreatments will lead to more efficient use of biomass. By combining multiple probes of structure, sensitive to different length scales, with molecular dynamics simulations, we reveal two fundamental processes responsible for the morphological changes in biomass during steam explosion pretreatment: cellulose dehydration and lignin-hemicellulose phase separation. We further show that the basic driving forces are the same in other leading thermochemical pretreatments, such as dilute acid pretreatment and ammonia fiber expansion.

Organization and flexibility of cyanobacterial thylakoid membranes examined by neutron scattering.

Background: In cyanobacteria, light harvesting and photosynthesis occur in the thylakoid membranes. Results: The distances between thylakoid membranes are correlated with the size of the phycobilisome antenna and change reversibly and rapidly upon illumination. Conclusion: Thylakoid membranes have a structural plasticity tied to the regulation of photosynthesis. Significance: Characterizing the structural changes in photosynthetic membranes is crucial for understanding light harvesting and photosynthetic productivity. Cyanobacteria are prokaryotes that can use photosynthesis to convert sunlight into cellular fuel. Knowledge of the organization of the membrane systems in cyanobacteria is critical to understanding the metabolic processes in these organisms. We examined the wild-type strain of Synechocystis sp. PCC 6803 and a series of mutants with altered light-harvesting phycobilisome antenna systems for changes in thylakoid membrane architecture under different conditions. Using small-angle neutron scattering, it was possible to resolve correlation distances of subcellular structures in live cells on the nanometer scale and capture dynamic light-induced changes to these distances. Measurements made from samples with varied scattering contrasts confirmed that these distances could be attributed to the thylakoid lamellar system. We found that the changes to the thylakoid system were reversible between light- and dark-adapted states, demonstrating a robust structural flexibility in the architecture of cyanobacterial cells. Chemical disruption of photosynthetic electron transfer diminished these changes, confirming the involvement of the photosynthetic apparatus. We have correlated these findings with electron microscopy data to understand the origin of the changes in the membranes and found that light induces an expansion in the center-to-center distances between the thylakoid membrane layers. These combined data lend a dynamic dimension to the intracellular organization in cyanobacterial cells.

Structure of star-burst dendrimers: A comparison between small angle x-ray scattering and computer simulation results

We investigated the generation dependent shape and internal structure of star-burst dendrimers under good solvent conditions using small angle x-ray scattering and molecular modeling. Measurements have been performed on poly(amidoamine) dendrimers with generations ranging from g=0 up to g=8 at low concentrations in methanol. We described the static form factor P(q) by a model taking into account the compact, globular shape as well as the loose, polymeric character of dendrimers. Monomer distributions within dendrimers are of special interest for potential applications and have been characterized by the pair correlation function gamma(r), as well as by the monomer and end-group density profile, rho(r) and rho(e)(r), respectively. Monomer density profiles and gamma(r) can be derived from P(q) by modeling and via a model independent approach using the inverse Fourier transformation algorithm first introduced by Glatter. Experimental results are compared with computer simulations performed for single dendrimers of various generations using the cooperative motion algorithm. The simulation gives direct access to gamma(r) and rho(r), allows an independent determination of P(q), and yields in addition to the scattering experiment information about the distribution of the end groups. Excellent qualitative agreement between experiment and simulation has been found.

Observations of structure development during crystallisation of oriented poly(ethylene terephthalate)

Abstract Two types of SAXS and WAXS experiments have been made using synchrotron radiation to observe the transformation from smectic to crystalline phases in oriented poly(ethylene terephthalate) (PET). In step-anneal experiments, PET was drawn slowly at 30 °C and then observed after annealing at 5 °C steps up to 100 °C. In the other experiments, time-resolved observations were made while drawing at 90 °C at rates up to 10 s−1. Up to 70 °C the WAXS data in the step-anneal experiments showed the smectic meridional reflection reducing in lateral width, indicating an increase in lateral long range order with annealing. Between 70 and 100 °C, there was a reduction in the intensity of the smectic reflection which correlated with an increase in the intensity of crystalline reflections. The SAXS from the step-anneal experiments showed an intense equatorial streak which has a correlation peak around 20 nm and which diminishes with annealing above 70 °C. It is concluded that this feature is a characteristic of the presence of the mesophase in oriented PET and is due to elongated domains of smectic mesophase with a length >75 nm and with an interdomain spacing of around 20 nm. Between 70 and 100 °C the SAXS data showed additional diffuse diffraction which correlated quantitatively with the crystalline phase and evolved from a cross-like appearance to a well resolved four-point pattern. The time-resolved drawing experiments were limited by the time resolution of the SAXS detector. They showed the same development of four-point diffuse SAXS patterns as was observed in the step-anneal experiments and a very weak equatorial streak. Differences in phase transformation kinetics between the two types of experiment are attributed to the different chain relaxation processes available under different conditions.

The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory

Small-angle neutron scattering (SANS) is a powerful tool for characterizing complex disordered materials, including biological materials. The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility. Here, the technical specifications and performance of the Bio-SANS are presented. Sample environments developed to address the needs of the user program of the instrument are also presented. Further, the isotopic labeling and sample preparation capabilities available in the Bio-Deuteration Laboratory for users of the Bio-SANS and other neutron scattering instruments at ORNL are described. Finally, a brief survey of research performed using the Bio-SANS is presented, which demonstrates the breadth of the research that the instruments user community engages in.

A microscopic look at the reinforcement of silica-filled rubbers

The deformed structure of silica-filled elastomers under uniaxial strain has been investigated using a combination of both small angle x-ray scattering and small angle neutron scattering methods. Using an extraction procedure and taking into account the two-phase nature of these polymer-based composites, the single chain scattering behavior as well as filler properties could be obtained uniquely on identical samples. For the first time the deformation of the rubbery matrix on the length scale of the network chain in a filled rubber could be determined and therewith the importance of matrix overstrain for the mechanical properties was estimated. Additionally, the determination of filler deformation and filler destruction presents microscopic details of the mechanisms of filler networking and the stress-softening Mullins effect.

Small-angle neutron scattering reveals pH-dependent conformational changes in trichoderma reesei cellobiohydrolase I: Implications for enzymatic activity

Cellobiohydrolase I (Cel7A) of the fungus Trichoderma reesei (now classified as an anamorph of Hypocrea jecorina) hydrolyzes crystalline cellulose to soluble sugars, making it of key interest for producing fermentable sugars from biomass for biofuel production. The activity of the enzyme is pH-dependent, with its highest activity occurring at pH 4–5. To probe the response of the solution structure of Cel7A to changes in pH, we measured small angle neutron scattering of it in a series of solutions having pH values of 7.0, 6.0, 5.3, and 4.2. As the pH decreases from 7.0 to 5.3, the enzyme structure remains well defined, possessing a spatial differentiation between the cellulose binding domain and the catalytic core that only changes subtly. At pH 4.2, the solution conformation of the enzyme changes to a structure that is intermediate between a properly folded enzyme and a denatured, unfolded state, yet the secondary structure of the enzyme is essentially unaltered. The results indicate that at the pH of optimal activity, the catalytic core of the enzyme adopts a structure in which the compact packing typical of a fully folded polypeptide chain is disrupted and suggest that the increased range of structures afforded by this disordered state plays an important role in the increased activity of Cel7A through conformational selection.

pH dependent self assembly of β‐amyloid(10‐35) and β‐amyloid(10‐35)‐PEG3000

Small angle neutron and x-ray scattering (SANS/SAXS) studies were conducted on the structure of the aggregates formed from both the truncated model peptide -Amyloid(10-35) (A10-35) and a block copolymer -Amyloid(10-35)-PEG3000 (A10-35-PEG) in D2O at pHs from 3.0 to 7.0. These studies indicate that A10-35 aggregates into rod-like particles (fibril) and their radii are strongly dependent on the pH of the solution. The fibril-fibril association in A10-35 solutions is less at pH < 5.6, but becomes larger at higher pH. A10-35-PEG also assembles into rod-like particles whose radius is larger by about 30 A than that for A10-35 fibril at pH 4.2, while it is about 23 A larger at higher pH. Contrast matching SAXS/SANS experiments that eliminate the coherent scattering from PEG reveal that PEG moiety is located at the periphery of the fibril. Also the mass per unit length of the peptide portion is similar for both A10-35 and A10-35 fibrils at pH 5.6. The mass per unit length of the rods from SANS provides key information on the packing of A10-35 peptides in the fibril.

Characterization of sol-gel-encapsulated proteins using small-angle neutron scattering.

Entrapment of biomolecules in silica-derived sol-gels has grown into a vibrant area of research since it was originally demonstrated. However, accessing the consequences of entrapment on biomolecules and the gel structure remains a major challenge in characterizing these biohybrid materials. We present the first demonstration that it is possible with small-angle neutron scattering (SANS) to study the conformation of dilute proteins that are entrapped in transparent and dense sol-gels. Using deuterium-labeled green fluorescent protein (GFP) and SANS with contrast variation, we demonstrate that the scattering signatures of the sol-gel and the protein can be separated. Analysis of the scattering curves of the sol-gels using a mass-fractal model shows that the size of the colloidal silica particles and the fractal dimensions of the gels were similar in the absence and presence of protein, demonstrating that GFP did not influence the reaction pathway for the formation of the gel. The major structural difference in the gels was that the pore size was increased 2-fold in the presence of the protein. At the contrast match point for silica, the scattering signal from GFP inside the gel became distinguishable over a wide q range. Simulated scattering curves representing a monomer, end-to-end dimer, and parallel dimer of the protein were calculated and compared to the experimental data. Our results show that the most likely structure of GFP is that of an end-to-end dimer. This approach can be readily applied and holds great potential for the structural characterization of complex biohybrid and other materials.