Dynamics on Cellulose Show Two Important Populations from Neutron Scattering and Simulations

Elastic Intensity Scans
Elastic intensity scans of dry and hydrated cellulose. Dashed lines denote inflection points in the curves at 220 and 260 K, the temperatures at which the surface water (nonfreezing) and interfibrillar water (freezing) become mobile in the hydrated cellulose sample, respectively. Inset illustration depicts water populations associated with cellulose. [Adapted from O’Neill, H., et al. “Dynamics of Water Bound to Crystalline Cellulose.” Sci. Rep. 7, 11840 (2017). [DOI:10.1038/s41598-017-12035-w]. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/). Curve colors were modified, and additional labels and inset added.]
Biomass pretreatment is necessary to make cellulose accessible to hydrolysis for conversion to biofuels. Understanding water’s role in cellulose reactivity will aid discovery of the underlying processes that change biomass morphology and reactivity during different pretreatment regimes for biofuels production. In this study, cellulose-water interactions were examined using neutron scattering supported by molecular dynamics simulation. The data show two distinct populations of water molecules—one tightly “bound” to the surface and the other interfibrillar and translationally mobile. Accurate models of hydration water in the cell wall can address fundamental questions about cellulose-water interactions. The mobility of the interfibrillar water is also important to enzyme and chemical attack and is distinct from the bound and bulk water.

O’Neill, H. M., et al. “Dynamics of Water Bound to Crystalline Cellulose.” Sci. Rep. 7, Article 11840 (2017). [DOI:10.1038/s41598-017-12035-w].

Instruments and Facilities Used: Deuterium labeling, neutron scattering, and molecular dynamics simulation; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer, at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source (SNS). Structural characterization using small-angle neutron scattering (SANS) CG-3 Bio-SANS instrument at High Flux Isotope Reactor (HFIR) facility of ORNL and X-ray diffraction (XRD) analysis combined with SANS. Wide-Angle X-ray Diffraction (WAXD) using theta-theta goniometer Bruker D5005 instrument; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer at ORNL SNS; Molecular Dynamics (MD) Simulations (GROMACS software and the CHARMM C36 carbohydrate force field); and TIP4P water model.

Funding Acknowledgements: Deuterium labeling, neutron scattering, and molecular dynamics simulation; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer, at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source (SNS). Structural characterization using small-angle neutron scattering (SANS) CG-3 Bio-SANS instrument at High Flux Isotope Reactor (HFIR) facility, ORNL, and x-ray diffraction (XRD) analysis combined with SANS. Wide-angle x-ray diffraction (WAXD) using theta-theta goniometer Bruker D5005 instrument; QENS; molecular dynamics (MD) simulations (GROMACS software and CHARMM C36 carbohydrate force field); and TIP4P water model. H.O’N., J.H., B.E., J.C.S., P.L. and B.H.D. support: U.S. Department of Energy (DOE) Genomic Science Program, Office of Biological and Environmental Research (OBER), DOE Office of Science, under Contract FWP ERKP752, for sample preparation and QENS studies. SANS studies on Bio-SANS by S.V.P. and V.U. support: OBER-funded Center for Structural Molecular Biology (CSMB) under Contract FWP ERKP291, using facilities supported by the Office of Basic Energy Sciences (OBES), DOE Office of Science. Molecular dynamics (MD) simulations performed by L.P. supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center, funded by DOE OBES, under Award DE-SC0001090. Research used resources of the National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231. E.M. support: Oak Ridge National Laboratory’s (ORNL) Spallation Neutron Source (SNS), funded by the DOE OBES Scientific User Facilities Division. Manuscript authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with DOE.

Brown Rot Fungi Reveal a New Approach for Biomass Conversion to Fuels and Chemicals

Brown rot fungi

(A) Brown rot fungi mushrooms, (B) SANS profiles, (C) SFG spectra of brown rot fungi–mediated cellulose deconstruction, and (D) AFM images of repolymerized lignin in brown rot cell walls. [(A) Wikimedia Commons, Zinnmann; (B) Authors; (C) and (D) From Goodell, B., et al. “Modification of the Nanostructure of Lignocellulose Cell walls via a Non-Enzymatic Lignocellulose Deconstruction System in Brown Rot Wood-Decay Fungi.” Biotechnol. Biofuels 10, 179 (2017). [DOI 10.1186/s13068-017-0865-2]. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/).]
A multimodal approach used in this study examined wood decay by the brown rot fungi, Gloeophyllum trabeum or Rhodonia placenta, that degrade wood using a chelator-mediated Fenton (CMF) reaction. Small-angle neutron scattering (SANS) showed changes in microfibril bundling and lignin structure during biomass breakdown. Complementary approaches, sum frequency generation (SFG) spectroscopy, X-ray diffraction, atomic force microscopy (AFM), and transmission electron microscopy (TEM) also contributed information on nanoscale structural changes in wood over time. Woods studied were southern yellow pine (Pinus spp.) and birch (Betula verrucosa Ehr.). The data support a degradation mechanism in which sugars released by non-enzymatic action diffuse from the cell wall,  facilitated by increasing the porosity of the cell walls. This is a paradigm shift in understanding the mechanism of brown rot fungal degradation.

Goodell, B., et al. “Modification of the Nanostructure of Lignocellulose Cell Walls via a Non-Enzymatic Lignocellulose Deconstruction System in Brown Rot Wood-Decay Fungi.” Biotechnol. Biofuels 10(1), 179 (2017). [DOI:10.1186/s13068-017-0865-2].

Instruments and Facilities Used: Small angle neutron scattering (SANS) – Bio-SANS at High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory (ORNL). Sum frequency generation (SFG) spectroscopy, broadband SFG system. Chelator-mediated Fenton treatments (CMF) and cellulase treatment. X-ray diffraction analysis (XRD) – PANalytical Empyrean diffractometer, The Netherlands, equipped with a Cu X-ray source. Attenuated total reflectance Fourier transform infrared analysis (ATR-FTIR): Nicolet 8700 FTIR Spectrometer (Thermo Scientific) equipped with a smart iTR diamond ATR unit, a KBr beam splitter, and a deuterated triglycine sulfate (DTGS) detector. Atomic force microscopy (AFM) of brown-rotted wood surfaces. Nanoscope IIIa AFM-Digital Instruments, Santa Barbara, California with three 5 µm scans. Transmission electron microscopy (TEM) – Philips CM12 TEM instrument (Philips, Eindhoven, The Netherlands); images recorded on Kodak 4489 negative film and the films subsequently scanned using an Epson Perfection Pro 750 film scanner.

Funding Acknowledgements: Dr. Zhu support: Chinese Forestry Industry Research Special Funds for Public Welfare Projects (#201204702-B2). Eastwood support: UK Natural Environment Research Council, award #NE/K011588/1. Daniel support: Formas Grant 2015-469. Support from ORNL-Proposal IPTS-12345/CG-3. Sum frequency generation (SFG) spectroscopy, x-ray diffraction (XRD), and infrared (IR) studies support: Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center, funded by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under Award Number DE-SC0001090. Pingali and O’Neill support: Biofuels SFA funded by the DOE Genomic Science Program, Office of Biological and Environmental Research (OBER), under Contract FWP ERKP752. Bio-SANS support: Center for Structural Molecular Biology, supported by DOE OBER under Contract FWP ERKP291. Neutron scattering facilities at Oak Ridge National Laboratory (ORNL) support: Scientific User Facilities Division, DOE OBES. Research also supported by U.S. Department of Agriculture’s (USDA) National Institute of Food and Agriculture (NIFA) and University of Massachusetts Amherst’s Center for Agriculture, Food and the Environment and the Microbiology Department: project # MAS00511.

Direct Measurement of Protein Dynamics In Vivo

quasi-elastic neutron scattering (QENS)
Labeling strategy for probing live cells using quasi-elastic neutron scattering (QENS). Graph: QENS spectra of uninduced cells (blue) and cells with expressed GroEL protein (red) in the deuterium oxide (D2O) buffer. [Reprinted with permission from Anunciado, D. B., et al. “In Vivo Protein Dynamics on the Nanometer Length Scale and Nanosecond Time Scale.” J. Phys. Chem. Lett. 8(8), 1899–1904 (2017). DOI: 10.1021/acs.jpclett.7b00399. Copyright 2017 American Chemical Society.]
Quasi-elastic neutron scattering was used to study the protein GroEL in vivo. Protiated GroEL was over-expressed in deuterated Escherichia coli cells by addition of protiated amino acids during induction of GroEL. The data showed retardation factors of ∼2 and ∼4 for the internal dynamics and global diffusion of the protein, compared to those of the protein in solution at the same concentration. Comparison with literature values suggests that the effective diffusivity of proteins depends on the length or time scale probed. Selective isotope labeling of biomolecules in cells opens up new lines of biological research using “in-cell neutron scattering” to extract information on dynamics of biomolecular systems that is unobtainable by other analysis techniques.

Anunciado, D. B., et al. “In Vivo Protein Dynamics on the Nanometer Length Scale and Nanosecond Time Scale.” J. Phys. Chem. Lett. 8(8), 1899–1904 (2017). [DOI:10.1021/acs.jpclett.7b00399].

Instruments and Facilities Used: Quasi-elastic neutron scattering, BASIS neutron backscattering spectrometer at Spallation Neutron Source at Oak Ridge National Laboratory.

Funding Acknowledgements: Qiu Zhang: technical assistance. Manuscript authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy (DOE Neutron scattering experiments at Oak Ridge National Laboratory’s (ORNL) Spallation Neutron Source (SNS) support: Office of Basic Energy Sciences (OBES) Scientific User Facilities Division, DOE Office of Science. Additional funding: Adaptive Biosystems Imaging project (ERKP851) and Center for Structural Molecular Biology (Project ERKP291) supported by DOE Office of Biological and Environmental Research (OBER).