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Dynamic Visualization of Lignocellulose

Integration of computer simulation with neutron scattering to examine the structure of lignocellulose.

Loukas Petridis, Sai Venkatesh Pingali, Micholas Smith, Riddhi Shah, Yunqiao Pu, Volker Urban, Barbara R. Evans, Hugh M. O’Neill, Art Ragauskas, Paul Langan, Jeremy C. Smith, Brian H. Davison

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Project Goals

Lignocellulose enzymatic degradation

Lignocellulosic biomass is recalcitrant to deconstruction and saccharification due to its fundamental molecular architecture and multicomponent laminate composition. A fundamental understanding of the structural changes and associations that occur at the molecular level during biosynthesis, deconstruction, and hydrolysis of biomass is essential for improving processing and conversion methods for lignocellulose-based fuels production. The objective of this research is to develop and demonstrate a combined neutron scattering and computer simulation technology for multiple-length scale, real-time imaging of biomass during pretreatment and enzymatic hydrolysis.



 We have extended our studies in small-angle neutron scattering (SANS) and in simulations beyond dilute acid pretreatment into other important or novel pretreatments, such as ammonia and ionic liquids. The results have confirmed and deepened the understanding of pretreatment effects on cellulose structure and lignin hydration and aggregation. 

We extended those studies by combining multiple techniques in characterizing biomass structure (Langan et al., 2014). We performed fiber diffraction and ex situ and in situ SANS and NMR experiments on steam explosion pretreatment of poplar to rationalize processes that drive biomass pretreatment. We have revealed the fundamental processes that drive biomass pretreatment by combining multiple probes of structure (SANS, NMR, crystallography), sensitive to different length scales, with MD simulations. We observed two processes, a hemicellulose-lignin phase separation and a dehydration of the cellulose fibril interface, that are common to multiple pretreatment methods (Langan et al., 2014; Pingali et al., 2014).  

We performed contrast variation SANS studies to establish (a) the scattering length density of the substrate necessary for determining the solvent ratio to contrast match substrate scattering and (b) the effect of D incorporation on biomass ultra-structure. A key result is that, while at first approximation, complex biomass is uniformly deuterated, there are measurable differences in D incorporation between carbohydrate and lignin. Lignin had a slightly lower D level, perhaps because of the longer biosynthetic pathways (Evans et al., 2014). 

In addition, we improved our ability to produce deuterated material at up to 100% for bacterial cellulose (He et al., 2014; Bali et al., 2013) and almost 40% for complex biomasses such as ryegrass (Evans et al., 2014). These studies also developed a novel technique to produce partially deuterated switchgrass (Evans et al., 2015) that is the first finding of a sufficiently deuterated perennial herbaceous biofeedstock to allow contrast matching SANS experiments. This material will be a great resource for future studies. 

Enzyme-cellulose interactions were explored using both simulations (Alekozai et al., 2014) and SANS to study Trichoderma reesei Cel7A in solution or bound to cellulose, suggesting that the catalytic core/carbohydrate binding module (CBM) distance contracts when bound to cellulose. SANS experiments obtained an ensemble of conformations of Cel7A enzyme during cellulose digestion. MD simulations of Cel7A were compared with SANS experiments to determine the solution structure of Cel7A. We also performed SANS of cellulase interaction with lignin. Thermochemical treatment causes lignin particles to associate, increasing the proportion of larger particles significantly; and the addition of Cel7A protein increases the population of discrete lignin particle sizes, implying the association of Cel7A enzyme with lignin aggregate particles. 

We performed structural studies of Populus tension wood (tension, opposite and control regions) using X-ray fiber diffraction and SANS. External forces such as bending tension in woody biomass cause the biological formation of larger fibrils with a more highly crystalline nature—an effect similar to thermochemical treatment of biomass, such as dilute acid pretreatment. These results have led to a strong recognition of the critical role of water (or other solvent) hydration in the structure and pretreatment of biomass (Lindner et al, 2013; Langan et al., 2014; Sawada et al., 2014; Petridis et al., 2014).

Research Highlights

More Dynamic Lignin is Controlled by Hydration and Thermal Processing and is easier to Valorize 

Segregation of Organosolv Solvents at the Cellulose Interface Influences Water Dynamics and Cellulose Deconstruction

Water dynamics on cellulose show two important populations from neutron scattering and simulations

Neutrons inform how to improve durability of forest products

ORNL TITAN supercomputer used to determine molecular mechanism of lignin’s adverse effect in biofuel production

Silane functionalization of silicon cantilevers with chip holder demonstrated for chemical force imaging of switchgrass

Switchgrass Produced with 34% D/H Substitution by Hydroponic Cultivation for Neutron Scattering Experiments

Integration of Powder Diffraction and Simulation Required for Accurate Determination of Cellulose Crystallinity

Five Papers in Cellulose Showcase Power of Neutron Scattering

Neutrons and Simulation Reveal Coupling of Dynamical and Mechanical Properties of Cellulose

BER Biofuels SFA: Patent Issued for "High-Throughput Reproducible Cantilever Functionalization"

Neutrons Probe Common Processes Responsible for Structural Changes during Biomass Pretreatment

Morphological Changes in Cellulose and Lignin Components of Biomass Occur at Different Stages during Steam Pretreatment

Effect of Deuteration on the Structure of Bacterial Cellulose

High-Performance MD Simulation of Lignin Aggregation on Cellulose Shows Stronger binding to Crystalline Regions

Effect of Amines on Disruption of Cellulose

Neutron Technologies for Bioenergy Research

Complex Molecular Architectures of Lignin Measured via Neutron Scattering

Simulation Analysis of the Temperature Dependence of Lignin Structure and Dynamics




53. Vural, D., Gainaru, C., O’Neill, H., Pu, Y., Smith, M. D., Pingali, S. V., Mamontov, E., Davison, B. H., Sokolov, A. P., Ragauskas, A. J., Smith, J. C., Petridis, L. “Impact of hydration and temperature history on the structure and dynamics of lignin.” (Green Chemistry, 2018, DOI: 10.1039/C7GC03796A)  

52. Smith, M. D., Cai, C.; Cheng, X., Petridis, L., Smith, J. C. “Phase-dependent solvation of xylan in tetrahydrofuran-water enables synergistic processing of lignocellulosic biomass.” (accepted for publication Green Chemistry). 


51. Vural, D., Smith, J.C., Glyde, H. “Determination of Dynamical Heterogeneity from Dynamic Neutron Scattering of Proteins.” (accepted for publication Biophysical Journal). 


50. Kumar, R., Bhagia, S., Smith, M. D., Petridis, L., Ong, R., Cai, C. M., Mittal, A., Himmel, M. E., Balan, V., Dale, B. D., Ragauskas, A., Smith, J. C., Wyman, C. E. "Cellulose-Hemicellulose Interactions at Elevated Temperatures Increase Cellulose Recalcitrance to Biological Conversion" (accepted for publication, Green Chemistry) DOI · 10.1039/C7GC03518G. 


49. Sawada. D., Kalluri, U., O'Neill, H., Urban, V., Langan, P., Davison, B., Pingali, S. V. (2018) "Tension wood structure and morphology conducive for better enzymatic digestion", Biotechnology for Biofuels, 11:44. 

48. Smith, J. C., Tan, P., Petridis, L. and Hong, L. "Dynamic Neutron Scattering by Biological Systems." (accepted for publication, Annual Review of Biophysics). 

47. Moyer, P., Smith, M. D., Abdoulmoumine, M., Chmely, S. C., Smith, J. C., Petridis, L., and Labbé, N. "Relationship between lignocellulosic biomass solubilization and physicochemical properties of ionic liquids composed of 3-methylimidazolium cations and carboxylate anions" (in pressJanuary 2018 · Physical Chemistry Chemical Physics 20(4), DOI 10.1039/C7CP07195G) 

46. Meng, X., Pu, Y., Sannigrahi, P., Li, M., Cao, S., Ragauskas, A. J. (2018) "The nature of hololignin." ACS Sustainable Chemistry & Engineering 6(1), 957-964. 

45. Smith, M. D., Cheng, X., Petridis, L., Mostofian, B., Smith, J. C. (2017) "Organosolv-Water Cosolvent Phase Separation on Cellulose and its Influence on the Physical Deconstruction of Cellulose: A Molecular Dynamics Analysis." Scientific Reports 7 (1), 14494. 

44. Evans, B. R., Bali, G., Ragauskas, A., Shah, R., O'Neill, H., Howard, C., Lavenhouse, F., Ramirez, D., Weston, K., Ramey, K., Cangemi, V., Kinney, B., Partee, C., Ware, T., and Davison, B. (2017) "Alleopathic effects of exogenous phenylalanine: A comparison of four monocot species." Planta 246 (4), 673 - 685. 

43. Meng, X., Evans, B. R., Yoo, C. G., Pu, Y., Davison, B. H., Ragauskas, A. J. (2017) "Effect of in Vivo Deuteration on Structure of Switchgrass Lignin." ACS Sustainable Chemistry & Engineering 5(9), 8004 - 8010. 

42. O'Neill, H.M.; Pingali, S.V.; Petridis, L.; He, J.; Evans, B.; Mamontov, E.; Hong, L. Urban, V.; Langan, P.; Smith, J.C.; Davison, B. (2017) "Dynamics of water bound to crystalline cellulose." Scientific Reports 7, Article number: 11840 [doi:10.1038/s41598-017-12035-w]. 

41. Goodell, B., Zhu, Y., Kim, S., Kafle, K., Eastwood, D., Daniel, G., Jellison, J., Yoshida, M., Groom, L., Pingali, S. V. O'Neill, H. (2017) "Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown-rot wood decay fungi." Biotechnol. For Biofuels 10:179 []. 

40. Pingali, S. V., Urban, V. S., Heller, W. T., McGaughey, J., O'Neill, H., Foston, M. B., Li, H., Wyman, C. E., Myles, D. A., Langan, P., Ragauskas, A., Davison, B., Evans, B. R. (2017) ACS Sustainable Chemistry & Engineering 5(1), 426 -435. "Understanding Multiscale Structural Changes During Dilute Acid Pretreatment of Switchgrass and Poplar." [DOI: 10.1021/acssuschemeng6b01803]. 

39. Sawada, D., Ogawa, Y., Nishiyama,Y., Togawa, E., Kimura, S., Langan, P. (2016) Crystal Growth & Design 16(6), 3345-3352. "Molecular interactions in an ɑ-chitin/hydrazine complex: Dynamic hydrogen bonds and improvement of polymeric crystallinity." 

38. Plaza, N. Z., Qian, S., Heller, W. T., Pingali, S. V., Jakes, J. E. (2016) Cellulose 23(3), 1593 - 1607. "Informing the improvement of forest products durability using small angle neutron scattering." 

37. Petridis, L., and Smith, J. C. (2016) ChemSusChem 9(3), 289 - 295. "Conformations of low-molecular weight lignin polymers in water." 

36. Evans, B. R., & Shah, R., "Development of Approaches for Deuterium Labeling in Plants", in Methods in Enzymology: Volume 565 Isotope Labeling of Biomolecules, ed. Kelman, Z., Chapter 10, Elsevier Ltd., Oxford, Great Britain (2015), pp. 213 - 243. 

35. O'Neill, H., Shah, R., Evans, B., He, J., Pingali, S. V., Chundawat, S. P. S., Jones, A. D., Langan, P., Davison, B. H., Urban, V., "Production of Bacterial Cellulose with Controlled Deuterium-Hydrogen Substitution for Neutron Scattering Studies" in Methods in Enzymology: Volume 565 Isotope Labeling of Biomolecules, ed. Kelman, Z., Volume 565, Chapter 6, Elsevier Ltd., Oxford, Great Britain (2015), pp. 123 - 146. 

34. Vermaas, J. V., Petridis, L., Qi, X., Schulz, R., Lindner, B., Smith, J. C. "Mechanism of Lignin Inhibition of Enzymatic Biomass Deconstruction" (2015) Biotechnol. Biofuels 8(1), 1-16. 

33. G. Bali, X. Meng, J.I. Deneff, Q. Sun, A.J. Ragauskas,; “The Effect of Alkaline Pretreatment Methods on Cellulose Structure and Accessibility in Milled Populus””, Chem. Sus. Chem., 8(2), 275-279 (2015)

32. I. Lee, B. Evans, G. Bali, M. Foston, A.J. Ragauskas,; “Silicon cantilever functionalization for cellulose-specific chemical force imaging of switchgrass””, Analytical Methods, 7, 4541 (2015)

31. B. Evans, G. Bali, M. Foston, A.J. Ragauskas, H.M. O'Neil, R. Shah, J. McGaughey, D. Reeves, C.S. Rempe, B.H. Davison ; “Production of deuterated switchgrass by hydroponic cultivation”, Planta 242(1), 215 - 222 (2015)

30. B. Lindner, L. Petridis, P. Langan and J. C. Smith; “Cellulose Crystallinity and Powder Diffraction Diagrams”, Biopolymers 203, 67-73 (2015)

29. L. Petridis, H. M. O’Neill, M. Johnsen, B. Fan, R. Schulz, E. Mamontov, J. Maranas, P. Langan and J.C. Smith “Hydration Control of the Mechanical and Dynamical Properties of CelluloseBiomacromolecules 15, 4152–59 (2014).

28. L. Hong, L. Petridis and J. C. Smith; “Biomolecular Structure and Dynamics: The View from Simulation”, Israel Journal of Chemistry 54, 1264-1273 (2014)

27. Q. Sun, M. Foston, D. Sawada, S.V. Pingali, H.M. O’Neill, H. Li, C.E. Wyman, P. Langan, Y. Pu and A.J. Ragauskas, "Comparison of Changes in Cellulose Ultrastructure During Different Pretreatments of Poplar”, Cellulose 21, 2419-31 (2014).

26. D. Sawada, L. Hanson, M. Wada, Y. Nishiyama and P. Langan, “The Initial Structure of Cellulose During Ammonia Pretreatment”, Cellulose 21:1117-1126, (2014).

25. A.J. Ragauskas, G.T. Beckham, M.J. Biddy, R. Chandra, F. Chen, M.F. Davis, B.H. Davison, R.A. Dixon, P. Gilna, M. Keller, P. Langan, A.K. Naskar, J. N. Saddler, T. J. Tschaplinski, G.A. Tuskan and C.E. Wyman, “Lignin Valorization in the Biorefinery”, Science 16, 709 (2014).

24. E. M. Alekozai, P. K. Ghattyvenkatakrishna, E. C. Uberbacher, M. F. Crowley, J. C. Smith and X. Cheng,“Simulation Analysis of the Cellulase Cel7A Carbohydrate Binding Module on the Surface of the Cellulose Iβ”, Cellulose 21(2): 951–971 (2014).

23. D. Sawada, Y. Ogawa, S. Kimura, Y. Nishiyama, P. Langan, and M. Wada, “Solid-Solvent Molecular Interactions Observed in Crystal Structures of Beta-Chitin Complexes”, Cellulose 21(2):1007-1014 (2014).

22. J. He, S.V. Pingali, S.P.S. Chundawat, A. Pack, A. D. Jones, P. Langan, B.H. Davison, V. Urban, B. Evans and H. O’Neill, “Controlled Incorporation of Deuterium into Bacterial Cellulose”, Cellulose 21(2), 927–936 (2014).

21. Y. Nishiyama, P. Langan, H. O'Neill, S.V. Pingali and S. Harton, “Structural Coarsening of Aspen Wood by Hydrothermal Pretreatment Monitored by Small- and Wide-Angle Scattering of X-ray and Neutrons on Oriented Specimens”, Cellulose 21(2), 1015–1024 (2014).

20. S. V. Pingali, H. M. O’Neill, L. He, Y. Nishiyama, Y. Melnichenko, V. S. Urban, L. Petridis, B. Davison, and P. Langan, “Morphological Changes in the Cellulose and Lignin Components of Biomass Occur at Different Stages During Steam Pretreatment”, Cellulose 21(2), 873–878 (2014).

19. H. Wang, G. Gurau, S. V. Pingali, H. O'Neill, B. Evans, V. Urban, W. Heller, and R. Rogers, “Physical Insight into Switchgrass Dissolution in the Ionic Liquid 1-Ethyl-3-Methylimidazolium Acetate”, ACS Sustainable Chemistry & Engineering (April 13, 2014) 1264–1269 (2014).

18. B. Evans, G. Bali, D. Reeves, H. O'Neill, Q. Sun, R. Shah, and A. Ragauskas, A., “Effect of D2O on Growth Properties and Chemical Structure of Annual Ryegrass (Lolium multiflorum)”, Journal of Agricultural and Food Chemistry 62(12), 2592–2604 (2014).

17. P. Langan, L. Petridis, H. M. O’Neill, S.V. Pingali, M. Foston, Y. Nishiyama, R. Schulz, B. Lindner, B.L. Hanson, S. Harton, W.T. Heller, V. Urban, B.R. Evans, S. Gnanakaran, A.J. Ragauskas, J.C. Smith and B.H. Davison, “Common Processes Driving the Thermochemical Pretreatment of Lignocellulosic Biomass”, Green Chemistry 16, 63–68 (2014).

16. B. Lindner, L. Petridis, R. Schulz and J. C. Smith; “Solvent-Driven Preferential Association of Lignin with Crystalline Cellulose Regions in Multimillion Atom Molecular Dynamics Simulation”, Biomacromolecules 14(10), 3390–3398 (2013)

15. D. Sawada, Y. Nishiyama, L. Petridis, R. Parthasarathi, S. Gnanakaran, V. T. Forsyth, M. Wada, P. Langan, “Structure and dynamics of a complex of cellulose with EDA: insights into the action of amines on cellulose”, Cellulose, 20 1563-1571 (2013).

14. G. Bali, M. Foston, B. R. Evans, J. He, A. J. Ragauskas, "The effect of deuteration on the structure of bacterial cellulose”  Carbohydrate Research, 347, 82-88 (2013)

13. P. Langan, B. R. Evans, M. Foston, W. T. Heller, H. M. O’Neill, L. Petridis, S. V. Pingali, A. J. Ragauskas, J. C. Smith,  B. Davison, “Neutron Technologies for Bioenergy Research”  Industrial Biotechnology, 8, 209 (2012)

12. S. E, Harton, S. V., Pingali, G. A. Nunnery, D. A. Baker, S. H. Walker, D. C.  Muddiman, T. Koga, T. G.  Rials, V. S. Urban, and P. Langan (2012) ACS Macro Lett.  1, 568-573, “Evidence for Complex Molecular Architectures for Solvent-Extracted Lignins.

11. Foston, M.B.; McGaughey, J.; O’Neill, H.; Barbara R. Evans, B.R.; Ragauskas, A.J - “Deuterium Incorporation in Biomass Cell Wall Components by NMR Analysis”, Analyst 137, 1090-1093 (2012)

10. Petridis, R. Schulz, J.C. Smith – “Simulation Analysis of the Temperature Dependence of Lignin Structure and Dynamics", J. Am. Chem. Soc. 133, 20277-20287 (2011)

9. Petridis, S.V. Pingali, V. Urban, W.T. Heller, H.M. O' Neill, M. Foston, A. Ragauskas, and J.C. Smith – Phys. Rev. E83, 061911 (2011), “Self-Similar Multiscale Structure of Lignin Revealed by Neutron Scattering and Molecular Dynamics Simulation.

8. S.V. Pingali, H.M. O'Neill, J. McGaughey, V.S. Urban, C.S. Rempe, L. Petridis, J.C. Smith, B.R. Evans, and W.T. Heller – (2011) J. Biol. Chem.286, 32801-32809, “Small-angle neutron scattering reveals a pH-dependent conformational change in Trichoderma reesei cellobiohydrolase I: Implications for enzymatic activity.”

7. M. Foston, C.A. Hubbel, A.J. Ragauskas – “Cellulose isolation methodology for NMR analysis of cellulose ultrastructure,” Materials 4, 1985-2002 (2011).

6. J.C. Smith, M. Krishnan, L. Petridis, N. Smolin – “Structure and dynamics of biological systems: Integration of Neutron Scattering with Computer Simulation,” Dynamics of Soft Matter (2011)

5. S.V. Pingali, V. Urban, W.T. Heller, J. McGaughey, H.M. O' Neill, M. Foston, D. Myles, A. Ragauskas, B.R. Evans – Biomacromolecules 11, 2329-2335 (2010), “Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass."

4. S.V. Pingali, V. Urban, W.T. Heller, J. McGaughey, H.M. O' Neill, M. Foston, D. Myles, A. Ragauskas, B.R. Evans – ActaCryst. D66, 1189-1193 (2010), “SANS Study of Cellulose Extracted from Switchgrass.

3. Foston, M; Ragauskas, AJ, “Changes in lignocellulosic supramolecular and ultrastructure during dilute acid pretreatment of Populus and switchgrass", Biomass & Bioenergy, 34:1885-1895 (2010).

2. Schulz, R.; Lindner, B.; Petridis, L.; Smith, J.C. – J.Chem. Theory Comput.5, 2798–2808 (2009), “Scaling of Multimillion-Atom Biological Molecular Dynamics Simulation on a Petascale Computer.”

1. Petridis, L., and Smith, J.C. – J. Comp. Chem. 30(3):457-67 (2009). “A molecular mechanics force field for lignin.”


This research (ORNL Biofuels SFA) is funded by the Genomic Science Program, Office of Biological and Environmental Research, U. S. Department of Energy.

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