Hemicellulose polymers both rigidly associate with cellulose fibrils and extend into the matrix environment where they exhibit significant molecular motion and can interact with lignin polymers in the secondary plant cell wall.Differences across monocot and eudicot plant species have been observed, such as variable substitution patterns on hemicellulose which can dictate cellulose hemicellulose association morphology for arabinose substitutions.The details of lignin structure are particularly challenging to analyze due to high heterogeneity and mobility, so partial extraction of the polymer is often necessary for assessment.Lignin is a polyphenolic network formed from the oxidative cross linking of the monolignols p-coumaryl alcohol , coniferyl alcohol , and sinapyl alcoholas well as Ferulic Acid.Lignin exists within the plant cell matrix, interfacing with both hemicellulose and cellulose.61 Solution-state NMR in combination with MS, which relies on swelling ball-milled plant cell walls with deuterated solvents, provides detailed information on the types, functionalization, and abundance of lignin linkages present.However, due to the necessity for drying, mechanical treatment, dissolution, or solvent extraction in these techniques, previous work does not report on recalcitrance in the intact secondary plant cell wall.NMR is inherently an atomic resolution technique,vertical farming hydroponic as the observed signals derive from nuclear spin magnetic moments located at precise locations in the molecules under study.

In contrast to solution-state NMR, which requires solubilization of the sample, solid-state NMR methods allow for analysis of intact plant tissues.The cost of implementing solid-state NMR limited early studies of the plant cell walls.Access to cost effective 13C labeling has contributed to the feasibility of understanding plant cell walls with solid state NMR.The requirement of NMR-active13C isotopes was a major hurdle for characterizations of native plant cell wall structure.13C has a low natural abundance and the cost of early efforts at isotope incorporation restricted their use.Relatively low13C enrichment enabled early studies on hardwood.The ability to detect the relative populations of rigid polymers was then applied to samples of pure cellulose and heterogeneous assemblies containing cellulose including paper products, cotton, wood chips, and pulp.While early X-ray diffraction studies demonstrated the crystalline nature of cellulose in plant cell walls,solid-state NMR measurements provided more detail, such as the pattern of hydrogen bond interactions responsible for the macroscopic shape of in situ cellulose fibers.A set of 1D cross polarization measurements was successfully applied to crystalline cellulose in birch and spruce biomass and offered the possibility of detecting exterior and interior cellulose components in macroscopic cellulose fibers.These straightforward 1D experiments were also useful for characterizing amorphous cellulose after ionic liquid processing of crystalline cellulose fibrils.The 2D cross polarized refocused Incredible Natural Abundance Double Quantum Transfer Experiment reports on directly bonded carbon atoms within polymers and has been useful for probing rigid structures, for example, resolving carbons and 5 signals of cellulose in Populus euramericana hardwood samples and characterizing the structure of amorphous cellulose.A breakthrough occurred when a highly efficient method of 13C incorporationwas coupled with multi-dimensional solid-state NMR to investigate the primary plant cell wall structure.This series of studies provided both a compositional and architectural description of the primary plant cell wall.

Hemicellulose-cellulose interactions were found to be much less prevalent in the primary plant cell wall than suggested by earlier models based on solvent extracted hemicellulose and enzymatic hemicellulose digestion studies.Furthermore, it was also revealed that the hemicellulose xyloglucan interacts mainly with the flat surfaces of crystalline cellulose fibers,expanding on the idea of xyloglucan associating, cross linking, and embedding into cellulose fibrils.Semiquantitative distance measurements recorded with the Proton Driven Spin Diffusion experiments substantiated the organization of cellulose, xyloglucan, and pectin in primary cell walls of both monocot and eudicot cell species.These advances in 13C enrichment allowed the use of advanced solid-state NMR approaches shaping the primary plant cell wall architecture and how secondary plant cell wall architecture could be approached with solid-state NMR.The strategy of 13C glucose feeding is not suitable to the study of the secondary plant cell wall because the plants need to be grown to relative maturity and complicated by respiration-dependent glucose synthesis.The development of less expensive growth chambers, utilizing 13C enriched carbon dioxide as the sole carbon source, which support the growth of plants throughout their life cycle, enabled the efficient incorporation of 13C isotopesin to plant tissues.Multidimensional solid-state NMR revealed significant differences in the dominant hemicellulose-cellulose contacts in different plant species.For example, in eudicot Arabidopsis thaliana , 2-fold screw conformations of hemicellulose xylan , dictated by even patterns of substitutions, enable a close association with crystalline cellulose.In contrast, in monocot sorghum, the high degree and irregularity of arabinose substitution patterns on xylan dictate a 3-fold screw conformation,enabling association with amorphous cellulose.Additionally, in softwoods, cellulose fibrils can be tethered by both xylan and mannan hemicellulose, increasing the strength of the plant cell wall.

For the sorghum case, limitations in biochemical techniques prevent the analysis of carbohydrate substitutions on xylan and the solid-state NMR measurements which can show the xylan-cellulose interaction that is otherwise unobtainable using other methods.Carbon dioxide 13C labeling and new applications of advanced solid-state NMR techniques have helped elucidate the structure of lignin in the secondary plant cell wall.Signal enhancement by dynamic nuclear polarization demonstrated that lignin directly bridges hemicellulose polymers and interacts strongly with cellulose fibers in uniformly labeled switch grass, highlighting the role of lignin in supporting the 3D organization of hemicellulose and cellulose.However, effective penetration of the DNP reagent into the plant cell wall for this signal enhancement required 15–20 min of milling,which could perturb native lignin structure.Direct polarization experiments utilizing PDSD performed on 13C enriched poplar stems highlight a potential avenue to probe lignin contacts and spatial proximities through selective excitation and magnetization transfer from lignin to other polymers,which provide support for the putative organization of lignin in poplar,switch grass,and Arabidopsis.Further development of selective excitation and other solid-state NMR methods to probe biomass with minimal sample manipulation have the potential to provide a more complete picture of the secondary plant cell wall structure and how established sample preparation methods influence that structure.Although a wide variety of solid-state NMR methods can be applied to highly 13C enriched plant tissues, two methods provide rapid and straightforward characterization of the polymer organization in the secondary plant cell wall.First,vertical planters for vegetables the INADEQUATE approach provides an avenue for the characterization of the polymers present within a secondary cell wall sample at relatively high resolution and can distinguish at least three populations of amorphous and crystalline cellulose, in addition to three populations of xylan.Second, 13C-13C recoupling methods, such as PDSD and Dipolar Assisted Rotational Resonance , report on the spatial proximity of cellulose, hemicellulose, and lignin.

Polymers free, dynamic, and in the plant cell matrix are captured by the refocused Incredible Nuclear Enhancement by Polarization Transfer experiment.Lower power proton decoupling is used in the rINEPT so polymers only with high intrinsic mobilityare detectable.The rINEPT experiment techniques share commonalities with solution state NMR experiments used to evaluate lignin content in deconstruction efforts and marks an upper limit of dynamic polymers which can be captured with solid-state NMR.These experiments provide a more complete picture of the polymers in the plant cell wall than has ever been obtainable before.In plants, cellulose fibrils have genetically and environmentally determined sizes so continuous fibrils change their direction and length resulting in nonuniform fibril orientations in plant tissues.Amorphous cellulose is important in the plant cell wall because they support cellulose fibril junctions so fibrils can change directions and adapt in tissues.For plant cellulose fibrils , only a fraction of the cellulose polymers have perfect hydrogen bonding patterns and order associated with crystalline cellulose.Crystalline cellulose is predicted to be more digestible in deconstruction by hydrolases for chemicals like HMF.Amorphous cellulose polymers in plant cellulose fibrils are associated with indigestible material and treated as a marker for recalcitrance.However there is still ambiguity regarding if the amorphous cellulose is recalcitrant due to being out of register cellulose within the polymer assembly.Considering enzyme digestion, the current consensus correlates the amorphous cellulose content within fibrils with recalcitrance,which can be correlated with a crystallinity index.Hemicellulose, the 1-O-4 linked linear polysaccharides contributing to the structural strength of the plant cell wall, interacts with both lignin and cellulose.The role of hemicellulose as a tethering component within the plant cell wall was first proposed in the primary plant cell wall structure and coheres with mechanical strength of plants provided by the secondary plant cell wall.Past deconstruction methods targeting hemicellulose resulted in higher recalcitrance.Even with ionic liquid digestion, early NMR studies with pulsed sequences show decreased lignin yields and increase in amorphous cellulose.Structural hemicellulose associating with amorphous cellulose fibril surfaces concerns recalcitrance when more amorphous cellulose surfaces form upon mechanical processing as predicted by milled cellulose fibrils in cotton.Structural hemicellulose also cross links with lignin upon plant maturity which greatly complicates digestion of roughly 60% of the secondary plant cell wall.In fact, hemicellulose-first deconstruction methods were largely abandoned due to high observed recalcitrance and supported the switch for deconstruction techniques to focus on lignin first extraction from the secondary plant cell wall.Finally, lignin is critical to the plant and plant development within all species,and high lignin content is associated with recalcitrance.High ratio of G/S lignin is attributed to greater heterogeneity and branching patterns within lignin networks and thus correlated with recalcitrance.Past correlations of recalcitrance outputs to the order in which polymers are digested have directed many deconstruction techniques to a “lignin first” model.Unfortunately, lignin also plays a vital role in plant water transport, pathogenic protection, and maturity; many mutations aimed at eliminating lignin are lethal to the organism.The complexity and insolubility of plant cell wall samples often requires heavy sample manipulation in deconstruction.However, whether recalcitrance is introduced in sample preparation of plant biomass conversion to bio-products is a complicated issue to address given the major discrepancies between lab and industrial processes.An immediate motivation to adopt more systematic approaches is the energy investments differing between the lab and industrial scale.This can become problematic in cases involving massive solvent extraction techniques and other preprocessing techniques as energy does not always scale from laboratorial to industrial settings.One example is the frequently used mechanical preprocessing at the lab scale which often proves to be too energetically expensive at the industrial scale.So, tracking assumptions and changes in the native plant cell wall structure behind the discrepancies is critical so that lab scale optimizations can benefit industrial applications.Mechanical preprocessing is commonly used to reduce biomass particle size to increase solvent accessibility and polymer solubilization.Lab scale vibratory ball-milling achieves this goal by rapidly vibrating a chamber containing lignocellulosic biomass with grinding balls.Importantly, past studies on the plant cell wall structure used mechanical milling to prepare samples for analysis,so the outcomes have been influenced by non-native interactions and contacts between these polymers.However, milling leading to recalcitrance is frequently reported during lignocellulosic biomass conversion efforts, impeding the efficiency of subsequent processing and separation steps.Common preprocessing sample preparations taken before specific deconstruction methods should be under investigation because of the potential for introducing wide spread recalcitrance.Mechanical preprocessing leading to recalcitrance is reported during lignocellulosic deconstruction pathways at the lab scale, which impedes the efficiency of subsequent steps in biomass processing.Milling induced recalcitrance could be due to the production of reactive lignin species promoting aberrant hemicellulose-lign in crosslinks as the lignin self-associates and condenses, resulting in polymers which may be less accessible for digestion.Additionally, increased amorphous cellulose content and exposed cellulose surfaces produced from milling cellulose fibrils5 could induce reorganization of hemicellulose-cellulose contacts due to their multiple modes of interaction in the native plant cell wall.Although the cost of applying milling to biomass as a technique on an industrial scale makes it energetically impractical to apply, the impact of potential recalcitrance induction during lab scale methods development could still influence efforts in developing deconstruction pathways and estimating their effectiveness.One recent study on milling cellulose fibrils offers potential insight into what happens to cellulose fibrils.Cellulose fibrils are nonuniformly oriented in the plant cell wall which results in irregular signal detection making some spectroscopic techniques challenging on intact material.Cellulose fibrils scatter light nonlinearly because crystalline cellulose belongs to a noncentro symmetric crystal group.Milling pure cellulose in cotton allowed for easier sample orientation which is important in the Ling et al.2019 study contrasting 13 different techniques to study crystallinity, including x-ray scattering techniques, vibrational spectroscopy and 1D CP solid-state NMR.In the study, Field-Emission Scanning Electron Microscopywas used to monitor sample morphology for cotton milled between 15–120 minutes at 30 Hz.