Lignocellulosic biomass is a composite structure with crystalline cellulose, hydrated hemicellulose, and lignin as major components. It has long been recognized as a potential lowcost and sustainable source of mixed sugars for production of biofuels and other value-added chemicals. Plants have evolved superb mechanisms for resisting assault on their cell wall structural sugars from the microbial and animal kingdoms, collectively known as biomass recalcitrance. These mechanisms are comprised of factors that are believed to contribute to the inefficiency of enzymatic hydrolysis of biomass. The lignocellulosic fine structure, i.e. the way cellulose, hemicelluloses, and lignin are bonding with each other, and how the lignocellulosic fine structure evolves during hydrolysis due to the molecular interactions between biomass and enzymes, is thus crucial for logistic and specific design of enzymes and processes to overcome the above factors that slow down the hydrolytic reactions. However, such urgently needed information is pretty much missing because direct detection of lignocellulose component conformation and distribution is NOT possible so far.
In this EAGER project, Investigators Bingqian Xu from University of Georgia and Wen Zhou from Michigan Technological University will employ a unique approach which is to combine the newly developed CBM functionalized AFM (atomic force microscope) technology with computational modeling to directly detect lignocellulose component conformation and distribution, thereby overcoming the long-standing technical difficulties in realizing the dynamics of lignocellulosic components (conformation and distribution) during the enzymatic hydrolysis. An EAGER grant would support this collaborated research to explore this proposed high-risk, high-reward project by getting the much needed data. The aim is a tool and methodology for selection and design of better enzymes and processes to overcome the biomass recalcitrance efficiently.
The significance of the proposed research lies in the ability (1) to study the lignocellulosic fine structure in nanometer scale with molecular recognition, (2) to construct the 3D structural image of biomass particle, and (3) to monitor the lignocellulosic fine structure dynamics in hydrolysis. The combination of experimental and computational modeling methods will potentially provide a new approach and evidence to tackle the unsolved lignocelluloses component conformation and distribution, offering molecular scale understanding of the lignocellulose hydrolysis process which could be critical in overcoming biomass recalcitrance. In addition, development of the technology will also add unique capabilities for single molecule studies in other biosystems to probe the biomolecules and their interactions.
Lignocellulosic plant biomass has been recognized as a potential low-cost, renewable and sustainable source of mixed sugars for production of biofuels and other value-added chemicals, because of its availability in large quantities and its reproducibility. Because of its extreme structural complexity, lignocellulosic biomass has evolved superb mechanisms for resisting assault on its structural sugars from the microbial and animal kingdoms, collectively known as biomass recalcitrance. Though tremendous efforts were made, little is known about such lignocellulosic fine structure, i.e. lignocellulose component conformation and distribution, on (external) surface and in interior of pretreated biomass. As a result, suitable techniques and instrumentations that can investigate lignocellulosic fine structure and its interactions with hydrolytic enzymes at the molecular level are in great need. We developed a novel Atomic Force Microscopy (AFM)-based single molecule recognition imaging and single molecule dynamic force spectrocopy (DFS) technique to image the chemical component of the fine structures of plant cell wall cellulose and study the single molecule affinity carbohydrate binding module (CBM) and crystalline cellulose intercations. First, we used the CBM3a-modified gold nanoparticles to determine the behavior of the cellulose-binding module on the natural cellulose surface. We observed that the GNP-CBM3a complexes bound to the cellulose surface and closely aligned with cellulose extension. The binding behavior was proved to be real after comparing the topographic and recognition images of the same crystalline cellulose exposed area as well as the force measurement done by dynamic force microscopy. Quantitatively, the unbinding force between the CBM3a and crystalline cellulose was determined. Then, we studied the carbohydrate-binding module and plant cell wall cellulose interactions using a family 3 CBM and a family 2 CBM on both natural poplar plant cell wall and extracted single crystalline cellulose. The CBM molecules are functionalized on a pre-coated AFM tip and the unbinding forces are measured by single-molecule DFS. Different from the widely used traditional bulk experiments, we determined several dynamic and kinetic parameters, such as reconstructed free energy, binding energy and bond lifetime constant based on the measured single molecule unbinding forces, which are able to illuminate the affinity of the CBMs to the natural and single cellulose surface from a totally different aspect. Generally, it was found that CBM3a molecule has a little higher binding efficiency and affinity than CBM2a molecule to both natural and extracted cellulose substrate and both of the CBMs have higher affinities to the natural cell wall cellulose compared to the extracted single cellulose. As one of the most important carbohydrate-protein interactions, the CBM-cellulose interaction studied by this technique will offer a radical approach to provide more detailed information for not only the plant cell wall degradation but for other single molecule interaction systems. Most recently, we determined plant cell wall structural and molecular level component changes after pretreatments. In this study, we determined the structural changes, specifically on crystalline cellulose, of natural, dilute sulfuric acid pretreated and delignified cell wall surfaces of poplar, switchgrass, and corn stover using AFM recognition imaging. Based on area percentage calculations, our results showed that 17-20% of plant cell wall surfaces were covered by crystalline cellulose before pretreatment and this coverage increased to 23-38% after dilute acid pretreatment under different temperature and acid concentrations. When the plant cell walls were pretreated with 0.5% sulfuric acid, the crystalline cellulose surface distribution of 23% on poplar, 28% on switchgrass, and 38% on corn stover was determined as an optimized result at 135°C. Compared to bulk component analysis, this method exhibits pronounced advantages in providing detailed surface information of plant cell walls. Intellectual Merit : The significance of this research lies in the ability (1) to study the lignocellulosic fine structure in nanometer scale with molecular recognition, (2) to construct the 3D structural image of biomass particle, and (3) to monitor the lignocellulosic fine structure dynamics in hydrolysis. The methods will potentially provide a new approach and evidence to tackle the unsolved lignocellulose component conformation and distribution, offering molecular scale understanding of the lignocellulose hydrolysis process which could be critical in overcoming biomass recalcitrance. Broader Impacts: The method we developed in this study can a very powerful tool in understanding and solving biomass recalcitrance, e.g. investigate various combinations of hydrolytic enzymes on hydrolysis, and study effects of different pretreatment methods on hydrolysis. Our own development of the technology will also add unique capabilities for single molecular studies in other biosystems to probe the biomolecules and their interactions, as is demonstrated by our study of a small peptide molecule (FX06) binding to heparin (protein-polysaccharide interaction). Besides, the interdisciplinary nature of the research attains additional educational scope through its ability to recruit women and students from traditionally under-represented groups due to the PI's participation in the well-developed outreach network of the University of Georgia’s College of Engineering and Nanoscale Science and Engineering Center.
