Characterization, Neutron Scattering, and Molecular Dynamic Simulation of the Lignin Carbohydrate Complex Structure and its Disruption
Authors:
Sai Venkatesh Pingali7* (pingalis@ornl.gov), Yunxuan Wang1, Riddhi Shah6, Xianzhi Meng1, Yun-Yan Wang2, Austin Conte1, Manjula Senanayake6, Yunqiao Pu3, Micholas Dean Smith4,5, Mitra Mazarei7, Rupesh Agarwal4,5, Shalini Jayaraman Rukmani5, Hong-Hai Zhang8, Ajay K. Biswal, Hugh O’Neill7, Arthur J. Ragauskas1,2,4, Brian H. Davison4
Institutions:
1Department of Chemical and Biomolecular Engineering, University of Tennessee–Knoxville; 2Department of Forestry, Wildlife, and Fisheries, Center for Renewable Carbon, University of Tennessee Institute of Agriculture; 3Biosciences Division, Oak Ridge National Laboratory; 4Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee–Knoxville; 5Center for Molecular Biophysics, Oak Ridge National Laboratory; 6Neutron Scattering Division, Oak Ridge National Laboratory; 7Department of Plant Sciences, University of Tennessee–Knoxville; 8Center for Nanophase Materials, Oak Ridge National Laboratory; 9Complex Carbohydrate Research Center, University of Georgia
URLs:
Goals
Recent work aimed at improving the conversion of biomass to advanced biofuels and bioproducts has highlighted the critical importance of solvent effects. These effects are important both in the efficient solvent-based deconstruction of biomass and in the product titer limitations of fermentations due to the solvent-based destabilization of microbial membranes. This Science Focus Area provides fundamental knowledge about how solvents alter the structures of plant cell walls and of microbial membranes. The overarching hypothesis is that knowledge of partitioning or binding of the solvent from the bulk phase to biomass or biomembranes can help predict maximal or minimal disruption. Solvents disrupt biological structures comprising amphiphilic molecules and polymers (e.g., membranes and biomass). Determining common biophysical principles of solvent disruption will lead to new understandings of how solvents affect the relevant structures. This information will help determine the ultimate microbial limits in tolerating specific solvents, as well as the eventual design of cosolvents best suited for pretreatment. Researchers integrate the power of world class neutron scattering capabilities and leadership class supercomputing facilities available at Oak Ridge National Laboratory (ORNL). These capabilities are complemented by expertise in biodeuteration and biomembranes at ORNL, plant cell wall chemistry at the University of Tennessee, and neutron scattering and membrane biophysics at the University of Cincinnati.
Abstract
Effective conversion of biomass remains challenging. The three major components— cellulose, hemicellulose, and lignin—form a recalcitrant lignin-carbohydrate complex (LCC) that must be fractionated for valorization. The team’s studies on the molecular structural changes underline two approaches to improve biomass conversion: pretreatment improvement and feedstock genetic engineering. In the first case, researchers describe the mechanism of action of Cyrene in solubilization and fractionation of lignin during thermochemical pretreatment. In the second case, researchers investigated LLCs in pectin knockdown transgenic switchgrass and model composites to gain insight into the molecular details of lignin–carbohydrate interactions. Overall, this comprehensive analysis furthers the understanding of the solvent effect during biomass fractionation and critical polymer interactions in plant cell walls that impact biomass recalcitrance.
Cyrene is the trademark name of dihydrolevoglucosenone, a biodegradable, non-toxic green dipolar aprotic solvent the Circa Group produces on a scale of 50 tons per year. Cyrene effectively extracted a significant amount of lignin from hardwood, herbaceous species, and even softwood at an aqueous acidic mild temperature of 120℃ (Wang et al. 2023). Nuclear magnetic resonance (NMR) revealed that the structure of extracted lignin was modified, correlating to the composition of the Cyrene co-solvent system.
The interactions between Cyrene and lignin were studied by molecular dynamic simulation (MD), revealing that Cyrene facilitated lignin solubilization and disrupted lignin aggregation. Cyrene also modified the cellulose fraction of the biomass. Small-angle X-ray scattering (SAXS) showed no lignin aggregation on the surface of microfibrils after pretreatment. However, small angle neutron scattering (SANS) showed the distances between cellulose microfibrils increased after Cyrene pretreatment then decreased to a level similar to untreated biomass after incubation with a dilute alkaline solution, suggesting the presence of Cyrene between microfibrils and its removal after alkaline incubation (Figure 1). This comprehensive analysis demonstrated the high potential of Cyrene co-solvent fractionation in extracting lignin and enhancing fermentable sugar yield by revealing the molecular interactions between Cyrene and LCCs.
Engineered plants with reduced pectin exhibit lower recalcitrance towards conversion to biofuels (Biswal et al. 2018), but complexes of pectin and lignin have not been confirmed as playing a role in recalcitrance. Researchers utilized a model composite system to investigate the effect of pectins on lignin polymerization (Shah et al. 2023). The lignin monomer coniferyl alcohol, protiated or deuterated, was polymerized in vitro by the hydrogen peroxide-horseradish peroxidase method in the presence of homogalacturonan, a linear pectin found in grasses. These composites were characterized by Fourier-transform infrared spectroscopy, solid-state NMR, SAXS, and SANS experiments. The lignin-pectin composites were compared to lignin synthesized without pectin and a physical mixture of pectin and lignin. Lignin particle sizes were smaller in the composites, and interconnected networks were formed. A unique ester bond was detected, supporting the existence of covalent bonds as well as hydrophobic interactions between lignin and pectin. These insights into the role of pectin in lignin deposition in the cell wall may inform improving biomass and its deconstruction for biofuels and bioproducts.
Image
Cyrene co-solvent fractionation effectively enhanced the fermentable sugar yield of recalcitrant softwood biomass pine after enzymatic hydrolysis. SAXS revealed changes in microfibrils during the process due to the Cyrene.
References
Biswal, A. K, et al. 2018. “Sugar Release and Growth of Biofuel Crops are Improved by Downregulation of Pectin Biosynthesis,” Nature Biotechnology 36(3), 249–57. DOI:10.1038/nbt.4067.
Shah, R. S., et al. 2023. “Evidence for Lignin–Carbohydrate Complexes from Studies of Transgenic Switchgrass and a Model Lignin–Pectin Composite,” ACS Sustainable Chemistry & Engineering 11(3),15941–50. DOI:10.1021/acssuschemeng.3c04322.
Wang, Y.-Y., et al. 2023. “Characterization and Molecular Simulation of Lignin in Cyrene Pretreatment of Switchgrass,” Green Chemistry. DOI:10.1039/D3GC02239K2.
Funding Information
This work was supported by the BER program, U.S. DOE under ERKP752 at Oak Ridge National Laboratory (ORNL). Small angle neutron scattering (SANS) performed at the Bio-SANS, and BER program Structural Biology Resource with the High Flux Isotope Reactor and Spallation Neutron Source, a DOE User Facility. The Oak Ridge Leadership Computing Facility provided an Innovative and Novel Computational Impact on Theory and Experiment award as did National Energy Research Scientific Computing Center under DOE-ASCR support. Biswal was supported by the DOE-BER-BRC Center for Bioenergy Innovation.