What happens during cell wall deconstruction insights from experimental and computational studies Paul Langan Biology and Soft Matter Division, Neutron Sciences Directorate, Oak Ridge National Laboratory, Tennessee. DOE BER ERKP752 PI: Brian Davison This work was supported by the Genome Science Program, Office of Biological and Environmental Research, U.S. Department of Energy under contract FWP ERKP752. This work used resources of the Center for Structural Molecular Biology and the Bio-SANS and GP-SANS neutron beam-lines which are operated by Office of Biological and Environmental Research and the Office of Basic Energy Sciences of the U.S. Department of Energy. ORNL is managed by UT-Batelle LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR2272.
Overview CHALLENGE: Biomass is recalcitrant to conversion HYPOTHESIS: By imaging the physical and chemical changes that occur during biomass degradation we can guide process improvement APPROACH: Combine multiple probes of structure sensitive to different length scales, with MD simulations to dynamically visualize biomass during pretreatment RESULT: We revealed fundamental processes responsible for the morphological changes in biomass during steam explosion pretreatment IMPACT: Guiding principles for new pretreatments and plant modifications that accelerate biomass conversion Molecular Dynamics Simulations of Genetically Modified Lignin Carmona et al. Submitted, 2014. Common Processes Drive the Thermochemical Pretreatment of Lignocellulosic Biomass Langan et al. Green Chemistry, 2014. Morphological Changes in Cellulose and Lignin Components occur at Different Stages during Steam Pretreatment, Pingali et al., Cellulose, 2014. Simulation Analysis of the Temperature Dependence of Lignin Structure and Dynamics, Petridis et al., J. Am. Chem. Soc., 133, 20277 (2011)
ORNL operates 2 neutron scattering user facilities High Flux Isotope Reactor (HFIR) Intense steady-state neutron flux and a high-brightness cold neutron source Spallation Neutron Source (SNS) Powerful accelerator-based neutron source U.S. Department of Energy user facilities: Unique capabilities available through peer review
Neutrons, electrons, X-rays and NMR are complementary probes of biomass structure Neutrons are highly sensitive to hydrogen which can be selectively replaced by deuterium (D) Neutrons provide unique H 2 O/D 2 O contrast variation highlighting different components of complex systems D-Labeled Cellulose Lignin H 2 O D 2 O/H 2 O D 2 O
Neutron Computed Tomography Adaptive Imaging Platform: US Department of Energy Office of Biological and Environmental Research 10 µm to 1 µm resolution on prototype IMAGING station CG1D at HFIR 5
6 1 nm to 1000 nm resolution can be accessed by Small Angle Neutron Scattering (SANS) methods
7 Atomic resolution can be accessed by Neutron fiber diffraction (crystallography) methods
We characterized the structure of a wood biomass Experiment Equatorial Meridional Intact chips from aspen trees (Populus tremuloides) X-ray fiber diffraction Neutron fiber diffraction in 100% D2O to enhance contrast Results microfibril bundle 40 Å 40 Å L 100 Å Cartoon model of aspen cell wall 8 X-ray work was performed at the University of Toledo s X-ray Center and ORNL s Center for Structural Molecular Biology SANS work was performed at the ORNL High Flux Isotope Reactor s Bio-SANS and GP-SANS instruments
We then steam pretreated the biomass Before After X-ray fiber diffraction Neutron fiber diffraction in 100% D2O Samples were pretreated at Georgia Institute of Technology Key Results: 9 Crystalline domains in cellulose elementary fibils increase in size from 2nm to 4nm WHY? Side-by-side stacking of cellulose elementary fibrils disappears WHY? Globular lignin aggregates appear WHY?
MD simulations explained increased crystallinity and the loss of side-by-side packing in cellulose Simulations were on the Hopper Cray XE6 supercomputer at the National Energy Research Scientific Computing Center Interpretation: 1 As the temperature is increased a kinetic dehydration barrier is overcome as core water is released to the surrounding matrix in a thermodynamically entropy driven process, causing the fibrils to coalesce into bundles with more confined, and therefore ordered, surface chains. When the temperature is brought back down to RT the fibril bundles reach a more thermodynamically stable dehydrated state.
MD simulations explained lignin aggregation and pore formation Simulations were on the Hopper Cray XE6 supercomputer at the National Energy Research Scientific Computing Center Cartoon model of pretreated aspen cell wall Interpretation: As the temperature is increased lignin transitions to conformations that give rise to fewer entanglements with hemicellulose, allowing a phase separation, with lignin aggregating into crumpled globules. When the temperature is brought back down to RT, the lignin globules collapse due to unfavorable entropy of lignin hydration compared to bulk. 1
Looking at samples before and after pretreatment outside of the steam reaction vessel leaves unanswered questions 160 C 30 mins 40 C Heating Holding Cooling 1
In situ neutron scattering and MD simulations revealed the sequence of morphological changes Equator SANS work was performed at the ORNL High Flux Isotope Reactor s Bio-SANS instrument using a specially designed pretreatment reaction cell. Meridian Time-resolved neutron fiber diffraction Key Results: 1. Heating: water is expelled from cellulose microfibrils as fibers coalesce at 80C; Holding & Cooling: Minimal further change 2. Phase separation of lignin and hemicellulose starts ~140C. 3. Lignin aggregates grow in size 4. Lignin aggregates on cellulose
Common processes drive drive changes in biomass morphology during pretreatment The compensation between entropy and enthalpy of hydration drives the cell wall components over kinetic barriers, destabilizing them, and causing morphological changes that render cellulose more accessible to enzymes However, these changes also make cellulose more recalcitrant to enzymatic degradation and cause lignin to stick to cellulose Conclusion Modified pretreatments and biomass that promote lignin phase separation and extraction, whilst minimizing cellulose dehyrdation will enhance biomass conversion e.g. MAKE LIGIN MORE HYDROPHOBIC OR HEMICELLULOSE MORE HYDROPHILIC or REDUCE Tg 1
MD Simulations of Genetically Modified biomass Key Results: 1. More hydrophobic lignin tends to collapse and interact more favorably with itself 2. Increase in hydrophobicity of lignin decreases its interaction with hemicellulose
Summary We combined different probes of structure sensitive to different length scales and MD simulations to image biomass We determined the physical and chemical changes that occur during biomass degradation By identifying the fundamental forces that drive these changes we provide guiding principles for modifying biomass and pretreatment to enhance biomass conversion 16 Managed by UT-Battelle for the U.S. Department of Energy
Acknowledgements Bioscience Division, ORNL Loukas Petridis Ben Lindner Jeremy Smith Brian Davison Art Ragauskas Lilin He Biology and Soft Matter Division, ORNL H. O Neill S. V. Pingali D. Sawada Volker Urban Leif Hanson Riddhi Shah Chemistry Division, ORNL Barbara Evans Institute of Paper Science and Technology, GIT Marcus Foston Chemistry Department, UT Leif Hanson CERMAV Yoshi Nishiyama This work was supported by the Genome Science Program, Office of Biological and Environmental Research, U.S. Department of Energy under contract FWP ERKP752. This work used resources of the Center for Structural Molecular Biology and the Bio-SANS and GP-SANS neutron beam-lines which are operated by Office of Biological and Environmental Research and the Office of Basic Energy Sciences of the U.S. Department of Energy. ORNL is managed by UT-Batelle LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR2272.
Neutrons have broader potential in biofuels research Developing neutron scattering technologies to provide the user community with fundamental information that is needed to drive rational design of biomass, enzymes and pretreatments to enable cost-efficient production of biofuels. Small Angle Scattering Imaging Fiber Diffraction Reflectometry Spectroscopy Crystallography A Cray XT5 simulation model of lignocellulosic biomass (lignin and cellulose) together with an artists impression of a cellulase enzyme hydrolyzing the cellulose. In bioenergy research we plan to obtain a complete physical description of biomass deconstruction, and to scale up using coarse-graining simulation methods and mesoscale neutron techniques-jeremy Smith
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