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Fall 2020

Understanding the structure of lignin, the way nature makes it

Investigating the nanostructure of lignin using experimental and computational approaches

Sintu Rongpipi

Multifunctionality in nature is derived from structural heterogeneity. Biological molecules and their assemblies are highly complex in structure and versatile in function. The well-defined structures of biological molecules enable living organisms to perform complex synthetic functions, producing a wide range of products—from simple small molecules to macromolecular assemblies. Traditional chemical approaches can seldom mimic the synthetic capabilities of living organisms (Nick McElhinny, S. A. and J. J. Becker (2014). Frontiers in chemistry 2: 24-24). The plant cell wall is an example of such an intricate structure synthesized by molecular machineries in plants, where they perform several important functions in plant growth, mechanics, intercellular communication, and defense against pathogens. The cell wall is a heterogeneous mix of several biopolymers which are large molecular structures consisting of smaller molecules called monomers and are produced by living organisms. Based on structure, composition, and function, the plant cell wall can be classified as being either the primary cell wall or secondary cell wall. Primary cell walls are thin, flexible yet strong, walls surrounding cells, which are growing and dividing; secondary cell walls are thicker and even stronger walls surrounding cells that have stopped growing (Cosgrove and Jarvis 2012). 

Secondary cell walls form the main constituent of biomass, a renewable and sustainable, energy-rich source of non-fossilized carbon. It has three main constituent biopolymers: cellulose, hemicellulose, and lignin. Cellulose and hemicellulose consist of sugar molecules as their monomeric units and are called polysaccharides, while lignin consists of monomers called phenylpropanoid alcohols. While cellulose and hemicellulose are transformed into biofuels, lignin is produced worldwide mostly as a non-commercialized waste product. The term “lignin” originates from the Latin word lignum meaning wood. In plants, lignin performs several important functions such as providing rigidity and hydrophobicity, promoting water and minerals transport, and defending against pathogens. Even though an understanding of the chemical composition of lignin began more than a hundred years ago, a complete understanding of its structure and composition is still not available. The structural heterogeneity of lignin likely both contributes to its amazing functional versatility, while also critically limiting its industrial applications. 

Lignin is the most abundant renewable resource that contains aromatics groups, which contain alternating single and double carbon-carbon bonds in their cyclic (ring-shaped) chemical structure. These aromatic compounds, commonly derived from petroleum, are the feedstocks for products such as plastics, paints, and additives in food and pharmaceuticals. It has been reported that the aromatics that make up lignin could be a cheaper alternative to existing petroleum-based raw materials in several products such as coatings, paints, plastics, resins (plasticizer), and liquid fuels. For example, artificial vanillin (vanilla extract) is an important product that can be derived from lignin; however, as of today, it has been synthesized mostly from petrochemicals (http://polymerdatabase.com). The aromatic monomers of lignin, called phenylpropanoids, are synthesized, transported, and polymerized by plants through a complex process called lignin biosynthesis. Plants have the capability to alter the quantity of phenylpropanoids in response to changing environmental conditions. For example, plants enlist defensive strategies during dry conditions by producing more substances for defending against insects, and concomitantly reduce lignin production. Such variability in biosynthesis makes research into the lignin structure particularly challenging, mostly in plants growing under different conditions. A deeper understanding of lignin structure and biosynthesis will enable design of lignin-like, lignin-based materials, and will enable engineering agricultural crops that have a modified lignin content that provides mechanical strength and protection against pathogens.

Researchers at the Center for Lignocellulose Structure and Formation (CLSF) Energy Frontier Research Center are working toward understanding the structure and biosynthesis of lignocellulose. They have addressed several questions regarding the structure of lignin and its interactions with polysaccharides in secondary cell walls through experimental and computational approaches.

Solid-state NMR reveals lignin-polysaccharide interactions in plant secondary cell walls

A molecular understanding of the structure of cell walls is crucial for developing lignocellulose as a sustainable resource. Nuclear magnetic resonance (NMR) spectroscopy is used to determine molecular structure. It works on the principle that when a molecule is exposed to an external magnetic field, each nucleus will feel the effect of a modified field, depending on the presence of neighboring electric charges—the nuclei and electrons. NMR elucidates the molecular structure of a compound by measuring the response of nuclei to the local magnetic field, composed of the external field plus the field induced by neighboring electrons. Based on the response, researchers can deduce the chemical environment of all nuclei in the molecule, which can help deduce structure, composition, and dynamics. 

Traditionally, cell wall molecular structure has been characterized through solution-state NMR, which requires complete or partial dissolution of cell wall constituents using organic or ionic solvents. This can potentially disrupt the native state and interactions of biomolecules, introducing ambiguities in the understanding of wall structure. Solid-state NMR (SSNMR) has been able to resolve this problem to some extent, because it allows for structural characterization of native hydrated plant cell walls at the molecular level. Researchers at CLSF used SSNMR with sensitivity enhancements provided by Dynamic Nuclear Polarization (DNP) to study the interactions between lignin and cell wall polysaccharides in three energy-relevant crops (maize, switchgrass, rice), and Arabidopsis thaliana, which is a model system for research in plant biology (Kang, Kirui et al. 2019). DNP-NMR is a rapidly developing technique that provides significantly higher sensitivity and reduced measurement time than conventional NMR.

Figure 1: Revised model of the secondary cell wall based on the DNP-NMR study. The model indicates that lignin and polysaccharides are phase-separated, and lignin forms nanosized hydrophobic domains. The model also proposes that cellulose aggregates are not coated by lignin, but rather they are separated and connected by xylan. These newly emerged structural aspects differ from contemporary views of the secondary cell wall in which lignin and xylan are well mixed and cellulose is directly coated by lignin

This study provided many insights into lignin-polysaccharides interactions within the secondary cell wall, as illustrated in Figure 1, that contradict the previously accepted model of lignocellulose structure. The findings from the study can be summarized into the following key takeaways: (1) lignin self-aggregates into highly hydrophobic nanosized domains as represented by the orange-colored regions in Figure 1; (2) lignin and xylan, a hemicellulose, are phase-separated, i.e., they are not well mixed as hypothesized earlier (orange-colored lignin regions are well separated from blue- and pink-colored xylan regions in Figure 1); (3) cellulose is not extensively coated by lignin, but rather is spaced and connected by xylan (red-colored cellulose fibrils have limited contact with orange-colored lignin and they are separated by the strands representing xylan in Figure 1); and (4) different conformations of xylan bind to cellulose and lignin—a flat xylan (pink-colored strands in Figure 1) binds to cellulose while a non-flat xylan (blue-colored strands in Figure 1) binds to lignin.

Molecular dynamic simulations reveal the relationship between the structure of lignin and the sequence of its monomers

Lignin has a highly variable molecular structure but not a well-defined primary structure, leading to ambiguities in structure-function relationships. Many studies have been able to correlate the chemical composition of lignin to its technological application. For example, a higher content of a specific lignin monomer has been associated with the higher processability of lignin as a raw material. The relationship between lignin 3D structure and its monomeric sequence, however, remains unclear. Researchers at CLSF used all atom molecular dynamics (MD) simulations to elucidate this connection (Rawal, Zahran et al. 2020). MD is a computer simulation technique that predicts the time evolution of a system of interacting particles at an atomic scale. It is easier to learn how a system works if we can watch it, and atomic-level computer simulations such as MD allow researchers to do just that. Atomic-level MD simulations provide the means for watching the motions of individual entities in a molecule.

Figure 2: Initial 3D models of lignin used for MD simulations. (a) Linear S/G-lignin, (b) branched S/G-lignin, and (c) C-lignin. G, S, and C monomers are shown in green, purple, and yellow, respectively. The branching point subunit in (b) is shown as spheres. (d) The Proximal radial distribution function (pRDF), gprox(r) quantifies the structure of water around lignin. The reduced height of the first peak for C-lignin (red) compared to linear and branched S/G-lignin (green and blue) indicates that it has weaker interaction with water.

Researchers involved in this work studied two types of lignin: (1) a heteropolymer (consisting of different monomer types as represented by the green and purple regions in Figure 2a) called S/G-lignin, and (2) a homopolymer (consisting of only one monomer represented by the yellow region in Figure 2b) called C-lignin. The shape and size of these lignin molecules with differing sequence of monomers were compared. The study found that a monomeric sequence did not affect the shape and size. They found, however, that C-lignin is smaller in size than S/G-lignin. This implies that lignin structure may be affected more by the content of monomers than the sequence of the monomers.

Such insights into the nanostructure of lignin and its interactions with other wall polymers are crucial for the advancement of lignin as a sustainable raw material. A comprehensive understanding of plant cell wall structure and biosynthesis will aid in the design of lignin-like and cellulose-like sustainable materials, and in engineering genetically modified crops that have desirable mechanical strength and protection against pathogens, leading to agricultural productivity.

More Information

Cosgrove, D. and M. Jarvis (2012). "Comparative structure and biomechanics of plant primary and secondary cell walls." Frontiers in Plant Science 3(204).

Kang, X., A. Kirui, M. C. Dickwella Widanage, F. Mentink-Vigier, D. J. Cosgrove and T. Wang (2019). "Lignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR." Nature Communications 10(1): 347.

Rawal, T. B., M. Zahran, B. Dhital, O. Akbilgic and L. Petridis (2020). "The relation between lignin sequence and its 3D structure." Biochimica et Biophysica Acta (BBA) - General Subjects 1864(5): 129547.

Acknowledgments

Kang et al. The authors thank Drs. Riqiang Fu and Zhehong Gan for their experimental assistance, Dr. Brittney Nagle for proving the maize coleoptile walls, Dr. Hui Yang for providing the cellulose model, and Dr. Carol Taylor for constructive suggestions. DJC, along with Drs. Nagle and Yang, were supported as part of the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences under Award No. DE-SC0001090. The National High Magnetic Field Laboratory is supported by NSF through NSF/DMR-1644779 and the State of Florida. The MAS-DNP system at NHMFL is funded in part by NIH S10 OD018519 and NSF CHE-1229170. This work was supported by the National Science Foundation (NSF) through NSF OIA-1833040.

Rawal et al. We acknowledge Dr. Jeremy C. Smith for numerous insightful discussions of lignin structure. This research was supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences under Award DE-SC0001090. This research used resources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US Department of Energy under Contract DE-AC05-00OR22725.

About the author(s):

  • Sintu Rongpipi is a PhD candidate in Chemical Engineering at The Pennsylvania State University. She is a member of the Center for Lignocellulose Structure and Formation (CLSF). Her research focuses on characterization of plant cell walls using synchrotron X-rays radiation.

More Information

Cosgrove, D. and M. Jarvis (2012). "Comparative structure and biomechanics of plant primary and secondary cell walls." Frontiers in Plant Science 3(204).

Kang, X., A. Kirui, M. C. Dickwella Widanage, F. Mentink-Vigier, D. J. Cosgrove and T. Wang (2019). "Lignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR." Nature Communications 10(1): 347.

Rawal, T. B., M. Zahran, B. Dhital, O. Akbilgic and L. Petridis (2020). "The relation between lignin sequence and its 3D structure." Biochimica et Biophysica Acta (BBA) - General Subjects 1864(5): 129547.

Disclaimer: The opinions in this newsletter are those of the individual authors and do not represent the views or position of the Department of Energy.