The original proportion of amorphous and crystalline cellulose before and after milling and 30–50% of sample crystallinity was lost after milling cotton for 15 minutes.Based on 1D CP solid state NMR measurements around 40% of crystallinity was lost and the initial crystallinity of the sample accounted for some inherent amorphous cellulose compared to x-ray scattering techniques.When pure cotton cellulose was milled crystalline cellulose fibrils showed loss in crystallinity and a consistent increase in amorphous cellulose, supporting mechanical preprocessing could induce recalcitrance.To test the current mechanical induced recalcitrance hypothesis in mechanical preprocessing, sorghum stems were subjected to lab scale vibratory ball-milling.In this study, stem tissue was used for initial experiments to allow for the highest concentration of secondary plant cell wall to increase the likelihood of observing recalcitrance with the greatest signal-to-noise ratio possible for solid-state NMR.Monitoring changes in sample morphology of the cellulose fibrils by FE-SEM is necessary because milling times vary depending on the biomass, sample quantity, and desired particle size.The time points include milling for 2 minutes at 30 Hz as common initial mechanical preprocessing time consistently executed for biomass18 and 15 minutes of milling at the same frequency to emulate longer milling times.Contrasting the effects of milling pure cellulose and milling cellulose in the plant cell wall also informed the selection of the 15-minute milling time point.Molecular changes in secondary plant cell wall polymers are probed with an initial set of solid state NMR experiments.The rINEPT was expected to selectively probe the highly dynamic polymers, lignin, and hemicellulose.
The CP-rINADEQUATE 66 probes directly bonded carbons in rigid components such as cellulose of the cellulose fibril and hemicellulose associated to cellulose fibrils.The CP-PDSD experiments were used to monitor the atomic cellulose morphologies using through space carbon-carbon correlations.Molecular recalcitrance markers include mobility and contacts of the dominant polymers by solid-state NMR experiments for polymers lignin, hemicellulose,vertical lettuce tower and cellulose.Monitoring markers of recalcitrance can be subdivided into each type of polymer.During the milling process crystalline cellulose is expected to convert to amorphous cellulose according to previous work on pure cellulose fibril structures of cotton by 1D NMR, FE-SEM, and vibrational spectroscopy.The resulting conformation change to amorphous cellulose within the plant cell wall may result in decreased access to crystalline planes of cellulose necessary for enzyme digestion.Assuming sample matter is conserved and no digestion occurs, proportions of crystalline and amorphous cellulose content in cellulose fibrils can be reported by NMR.5 Unlike vibrational spectroscopy where there is high signal overlap and sample crystallinity indexes afforded by x-ray diffraction, NMR contains well separated signatures for amorphous and crystalline cellulose.Cellulose polymers on the dehydrated interior, center, and more hydrated interior cellulose polymers of the cellulose fibril have distinguished chemical shifts over the last 20 years of experiments and specifically identified for sorghum.24 In 2D solid-state NMR experiments plant cell wall polymers compromise over a third of biomass and most of the signals due to the 13C labeling technique.When examining the plant cell wall in the spectra the carbon neutral region of polys accharides and lignin is 120–60 ppm and 170–110 ppm, respectively.Tracking proportional intensity changes of amorphous and crystalline cellulose distinguished by their chemical environments is possible.
Cross Polarized NMR experiments will select for the rigid polymers as the experiments are able to spectroscopically separate the rigid portions of the plant cell wall including the cellulose fibrils.CP based solid-state NMR experiments are more efficient for spins stagnant in spacepolymers of the plant cell wall.Cellulose fibrils are solid structures which can be probed with CP based solid-state NMR.Hemicellulose rigidly associated to cellulose in cellulose-hemicellulose interactions would also be selected for using CP experiments.In the context of the plant cell wall, cellulosehemicellulose interactions do vary based on the morphology of cellulose,so recalcitrance could also occur by additional amorphous cellulose-hemicellulose interactions common in grasses such as sorghum.Hemicellulose signals would then increase in their rigid signal intensity according to CP experiments and decrease in signal intensity from the highly dynamic signals.The highly dynamic signals captured in the rINEPT include 1H-13C correlations in the 2-D experiment and lignin with the plant cell wall.Lignin reorganization could cause recalcitrance in a variety of ways.One observable way would be cross-linkages forming due to mechanical activation ligninarabinose-xylan.The ether signals within lignin branching and lignin-hemicellulose cross-linkages are captured within the rINEPT experiment.Lignin condensation, aggregation, and cross linking noted in recalcitrance means highly dynamic lignin content is lowered , resulting in an expected decrease in signal intensity in the rINEPT.Digestible hemicellulose and cellulose would be reduced as polys accharides are trapped as lignin condenses in the secondary plant cell wall.Based on previous 13C evident methods and solid-state NMR analysis, the molecular evaluation of secondary plant cell wall architecture is feasible for bio-product relevant crops such as sorghum.This research project aims to identify where recalcitrance occurs in the deconstruction pathway and identify markers of recalcitrance using 2D solid-state NMR.The first step of the deconstruction pathway is investigated for mechanically induced recalcitrance for 13C labeled sorghum.How the heterogeneous secondary plant cell wall changes from preprocessing needs to be carefully approached.This work primarily focuses on intensity changes of unambiguous signals within the established sorghum native plant cell wall to circumvent misassignment of polysaccharide non-ambiguous peak changes which shift to ambiguous, overlapped chemical shifts during deconstruction.
Gao et al.2020 is used as a reference for secondary plant cell wall polymer chemical shift assignments confirmed in sorghum from previous literature which relied on extracted polymers,computational verification,and previous solid-state NMR9,on the architecture of the plant cell walls.Additional structural information can be confirmed from chemical shifts using computation, allowing for polymer morphological changes to be assessed based on changes in monomer orientation.Future work using data collected in this work could offer greater insight into polymer morphology changes.Given the heterogeneity of the plant cell wall samples,vertical grow rack system examining the recalcitrant markers would narrow the relevant chemical shift and chemical shift changes by computation as well as contrast chemical shifts of extracted polymers.Chemical shifts can support changes in polymer environments when peaks are well dispersed and unambiguous within a 0.5 ppm.For example, accessible polymers are expected to be more hydrated and have chemical shifts downfield.As cellulose fibrils are broken down into more disperse micro-fibrils or cellulose substructures, as discussed in Figure 2C, the chemical shifts of amorphous and crystalline cellulose may be expected to move downfield.Additional hemicellulose-cellulose association on hydrophilic surfaces of cellulose polymers would have chemical shifts potentially shift upfield for both polysaccharides.Upon the hemicellulose crosslinking to lignin may also result in chemical shift changes downfield for hemicellulose due to deshielding from bonding with the conjugated heteroaromatic lignin.However, recalcitrant organization of lignin trapping hemicellulose and cellulose in self-aggregation would reduce surface water may have chemical shifts moderately upfield compared to the expected literature values in sorghum based on Gao et al.2020 assignments of the secondary plant cell wall.These are generally anticipated chemical shift changes in polymers which is somewhat challenging given signal overlap of polysaccharides.In this work changes in chemical shift after mechanical preprocessing are highlighted by 2D integration of chemical shifts found in the control.Integration allows for minor changes or gradual changes in the plant cell wall to be assessed, increasing the ease of interpreting reasonable chemical shift changes upon preprocessing.Sorghum was grown hydroponically and then transferred to a growth chamber for carbon dioxide 13C labeling until they reached 16 weeks by collaborators Yu Gao and Jenny Mortimer as described in Gao et al.2020.The sorghum was incorporated with 92% 13C according to elemental analysis and are physiologically normal organisms.Upon harvest, the plants were flash frozen in liquid nitrogen, divided by tissue type , and cryogenically stored for later studies.
Samples were transported on dry ice between labs and stored at −80 °C for the duration of the study.Cut stems were milled using a Retch MM400 in a 10 mL zirconium grinding jar with two 10 mm zirconium beads at 30 Hz.For the 2-minute milling time point, 600 mg of cut stems were milled for a single 2-minute interval.For the 15 minute milling period, 600 mg of cut stems were milled in cycles for 5 minutes followed by 5 minute resting periods in accordance to relevant lab scale milling procedures from previous literature.After the milling period, the samples were divided into several cryogen vials and flash frozen in liquid nitrogen before storage in the −80 °C freezer.Sample Morphology Tracked with FE-SEM FE-SEM was employed to check sample morphology, specifically cellulose fibril morphology structures after milling.A Hitachi S-4100 T Scanning Electron Microscope was used to collect high resolution images for the control and milled samples using a procedure detailed in Zheng et al.2020.98 A 10 nm gold coating was sputter coated onto control and milled sorghum samples with a Cressington 208hr Sputter Coater.FE-SEM images were collected at a working distance of 15–6 mm with acceleration voltages ranging from 2–10 kV to achieve optimal spectra resolution.Atomic Resolution of Sorghum Stem Secondary Plant Cell Wall by MAS-ssNMR NMR experiments were performed on a Bruker 500 Solids NMR Spectrometer , with a Bruker 4 mm MAS probe and at a MAS speed of 10 kHz at the UC Davis NMR Facility.All samples were shimmed using a water sample file on the instrument.Two channels were utilized on the 500 MHz instrument, one was set to SFO2 500.0305 MHz for the proton and the other was set to 125.7445782 MHz for 13C.Spinal 64 1H decoupling was used.Decoupling powers were optimized for 1H radio frequencies of 71 kHz.The radio frequency power for CP on the carbon for the 1D and 2D NMR was a matched between protons and carbons at the 10 kHz MAS spinning frequency.32 Optimization of the 1H and 13C RF pulse lengths for each sample were obtained by locating nulls a carbonyl signal around 100 ppm in a 1D CP experiment with 4 scans with parameters similar to the 1D CP in Table 1.An 1-13C L-alanine sample was used to check instrumentation and calibrate chemical shifts on the TMS scale using a 1D CP experiment prior to data collection on the plant samples.99 Beyond these commonalities, parameters for the experiments applied to all the control and milled samples are summarized in Table 1.Samples were packed into 4 mm zirconium rotors with two 2 mm Teflon discs cut from Teflon tubing on the top and bottom to center the 13C sample.All data collection occurred at room temperature.Experiments on control stems revealed the total time of sample viability in the spectrometer to be approximately 52 hours and this was tracked for all experiments by periodic the collection of 1D CP and DP spectra for all sample data collections.The 2D was collection is divided into two sets of experiment collection per experiment as summarized in Table 1 and added together in NMR pipe for processing.NMR data were processed in NMRpipe with Gaussian line broadening applied before the Fourier Transform.100 Spectra were plotted in Sparky101 and formatted in Corel Draw 2019.A quantitative 1D DP experiment was employed to properly scale data on the milled and control samples for integration.Parameters of the quantitative 30 s 1D DP experiments were like 1D-DP experiment in Table 1 but the recycle delay was adjusted to 30 second recycle delay to assess the 13C content within the control and the milled stem samples.After data collection samples were stored at −80 °C in their respective 4 mm rotors for later use in the 1D-DP to quantitatively scale the sample loading of the samples which varied in consistency.The 30 s 1D-DP allowed for integrations across all experiments for each sample to be scaled for sample load which varied due to the consistency variation in the cut and milled samples.Milled samples were flash frozen before NMR data collection without changes to the plant cell wall.Cut stems were thawed for 3.5 hours based on the appropriate milling time for DMSO gel swelled plant cell walls of grasses outlined for 600 mg of material in Kim and Ralph 2010.Experiments in Table 1 and a DP-rINADEQUATE show no detected changes.The DP-rINADEQUATE had the same experimental parameters outlined in the CP-rINADEQUATE summarized in Table 1 with the cross polarization removed and the 13C nuclei directly irradiated.This meant samples could be milled, flash froze, and rethawed for data collection.