Examining the reorganization of the secondary plant cell wall polymers due to mechanical preprocessing is important for the development of the plant cell wall model and effective utilization of biomass without recalcitrance.Conversion from plant biomass to bio-product often necessitates mechanical preprocessing in deconstruction methods; milling times vary but can be as short as 2 min and can exceed 4 hours.Milling of stem tissue at 30 Hz for 2 min was selected to allow direct comparison to DMSO swelling studies employing the same milling time.Typically plant cell wall samples are milled at 30 Hz for 2 minutes at 30 Hz followed by up to 24 hours of milling at 10 Hz depending on the amount of material.As a result, these experiments report on cell wall structure after reorganization of the plant cell wall polymers that occur during mechanical preprocessing.For example, solid-state NMR measurements on maize biomass after mechanical and solvent processing methods support lignin association with the surface of hemicellulose coated cellulose fibers in the cell wall,61 a different result than those obtained from recent solid-state NMR measurements on less processed grass and other plant species biomass.CO2 labeling is challenging for large mature plants such as Poplar trees given labeling chambers would need to adapt over the life cycle of the organism,vertical farming companies and there are few commercial sources.In this current study, the results of mechanical processing on 13C sorghum data collected at common laboratory milling times are available for native and milled stems to contrast the tissue with the highest amount of secondary plant cell wall to boost sensitivity solid-state NMR recalcitrance markers.
Comparisons are limited to changes in signal intensity signal through the integrated comparison of peaks found in the control to the milled samples for the CP-PDSD experiment, CP-rINADEQUATE, and the rINEPT.Polymers are evaluated across the secondary plant cell wall structure by combining the CP based experimental observations which examine highly rigid components and the rINEPT highly dynamic components of the sample.This current work also highlights initial characterization of highly dynamic lignin and hemicellulose polymers of the plant cell wall matrix in the rINEPT for the first time.However, the sorghum stems milled for 15 minutes will be compared to previous work by Ling et al.2019 which shows that cotton crystallinity of cellulose decreases by an average of 40% over 13 techniques.This is important because cotton is a naturally pure form of cellulose 5,38 and results from milling cellulose in the secondary plant cell wall can be observed in the sorghum stems milled for 15 minutes.For simplicity, the results are ordered by polymer class presented in Figure 2B: cellulose fibrils, structural hemicellulose, and lignin.FE-SEM images of the control and milled samples in Figure 6 show the morphological structure of the cellulose fibrils in their bundled cylindrical form as lines.Cellulose fibrils are typically on the order of 1-2 μm in diameters for a scale reference,which is on the order of some milling efforts of softwood “fines”.In Figure 6A the cellulose fibrils are shown in their bundled structure to the right in Figure 6B of the stem vasculature which can be seen as the large main cylindrical shape with thin cellulose fibrils forming lines across.After milling, the vascular structures comprising the stem are lost , this makes sense as the macroscopic stem structure is homogenized into a paste upon milling.However, the cellulose fibril structure remains after 2 minutes and 15 minutes of milling.Contrasting the control and stems milled for 2 minutes at 30 Hz, the individual cellulose fibers within the sample are largely similar, with a slightly rougher texture.After 15 minutes of milling stems at 30 Hz, there is noticeable fraying of the cellulose fibril bundles within Figure 6E, the thinner cylindrical lines appear to be consistent with microfibrils.
Very different results appear after milling cellulose fibrils for 15 minutes at 30 Hz in this study with previous work on milled cotton.Here for sorghum, more intact fibril morphology is maintained when milling the cellulose fibrils within a second plant cell wall.In the Ling et al.2019 study, cotton cellulose was fractured to the point microfibril structures were obscured and fibril chunks remained.There was qualitatively less severe cracking on the surface of fibrils in Figure 6E in the plant cell wall sorghum sample in this study and general fibril shapes are still apparent.In this current study, initial morphology of the intact cellulose fibrils in the plant cell wall was approached with FE-SEM and other techniques which may be considered for future evaluation of cellulose fibril structure within the heterogeneous plant cell wall during deconstruction for sorghum stems.Other techniques may yield further information related to recalcitrance due to lowered accessible cellulose fibril surfaces available for digestion.In the future, one class of attractive microscopies is vibrational microscopy for verifying cellulose including crystalline and amorphous cellulose.However, implementing these techniques suffer from the complexity of an intact plant cell wall and cellulose fluorescence.The benefit of microscopy over spectroscopy for assessing cellulose in the plant cell wall is the arrangement of the polymer in cellulose fibrils which vary in orientation and direction so techniques which can focus on one cellulose fibril at a time are favorable.Techniques such as Confocal Raman Spectroscopy with a 785 nm or 1064 nm lasing source or AFM-IR would both require optical arrangements suitable for detection of cellulose signals between 3000-400 cm-1 for informing on the cellulose fibril structure and on lignin or hemicellulose on cellulose fibril surfaces relevant for recalcitrance.
Vibrational microscopy would also have the advantage of confirming cellulose as the cellulose fibril structure is obscured in deconstruction.However for the scope of this current study includes FE-SEM structures were confirmed using literature98 and the assertion of cellulose predominance in the plant cell wall.For sorghum secondary plant cell walls subjected to vibratory milling, recalcitrance would be supported by the correlated decrease of crystalline cellulose and an increase in rigid amorphous cellulose.Such details can be extracted experimentally on the cellulose fibril structure using 2D solid-state NMR.Molecular insights specific to the constitution and state of the cellulose fibrils was assessed with CP-PDSD experiments with a 1500 ms and 30 ms mixing times to assess crystallinity.Peaks in the CP-PDSD were assigned using previously characterized peaks for polymers in the sorghum secondary plant cell wall Gao et al.2020and signals consistent with the CP-rINADEQUATE experiment for each dimension.The CP based experiments filter for more rigid components of the secondary plant cell wall because the 1H-13C magnetization transfers are more efficient for rigid spins.The mixing times of the CPPDSD has a proportional timescale to the distance of the spins between 13C-13C through space.The downside of the experiment is the broader line shapes due to heterogeneous line broadening from spins in multiple orientations coupling at similar frequencies,vertical garden indoor but crucial information about the cellulose fibril morphologies can still be obtained.The 1500 ms mixing period for the 1500 ms 2D CP-PDSD experiment reports on the larger cellulose structure along fibrils and between fibrils.The 30 ms CP-PDSD 30 experiment has a mixing time of 30 ms where there is enough time for the magnetization transfers to pass between the carbons within the glucose sugar of the monomer of the cellulose fibrils in Figure 8.First examination of the cellulose fibril shows magnetization transfers between D-glucose monomers of cellulose polymers in the 1500 ms CP-PDSD experiment.Cellulose carbon 1 to carbon 2 transfers have the same chemical shifts across both amorphous and crystalline cellulose.
The reductions in overall signal intensity considering sample load is negligible after 2 minutes of milling and >88% after 15 minutes of milling.This makes sense as cellulose fibrils appear to be broken down into smaller microparticle fragments.According to the FE-SEM images there may be fewer dipolar coupling-based magnetization transfers available along and between cellulose polymers after 15 minutes of milling.The proportional intensity changes between amorphous and crystalline cellulose signals of carbon 1 to 4 and carbon 1 to 6 magnetization transfers were assessed in the CP-PDSD experiments to identify the conversion of crystalline cellulose to amorphous cellulose.The proportion of the crystalline to amorphous cellulose for the glucose carbon 1 to carbon 4 transfer appears to decrease more for the 2-minute milling period, to 99.70 ± 1.59% and 82.17 ± 0.88% respectively of the relative peak intensity before milling.The signal intensity for the crystalline cellulose and amorphous cellulose appear to be nearly equal for the glucose carbon 1 to carbon 4 after 15 minutes of milling.The cellulose carbon 4 is of particular relevance as the amorphous cellulose has a chemical shift around 84 ppm and the crystalline cellulose has a chemical shift around 89 ppm.The isolation of cellulose carbon 4 in solid-state NMR spectra makes it a more reliable marker for amorphous and crystalline cellulose because they have less overlap than other peaks.After 2 minutes of milling the crystalline cellulose content appears higher than the amorphous cellulose content and the trend appears to also hold true for the 15 minute period.The reduction of amorphous cellulose signal may be due to amorphous cellulose becoming more mobile, resulting in less efficient CP transfer necessary for the 1500 ms CP-PDSD experiment.However, Ling et al.2019 found that even within the 1D CP experiments a conversion from crystalline to amorphous cellulose was observable as part of their prediction: crystalline cellulose within cellulose fibrils becomes amorphous upon milling.The conversion of crystalline to amorphous cellulose observed after milling was not consistently observed with sorghum.The ratio of crystalline cellulose to amorphous remains the same in 1500 ms CP-PDSD experiments as larger fibril structures are broken down in the milling process.The proportional intensities of crystalline and amorphous cellulose signals for carbon 1 to 4 remained the same after milling consistently for 2 minutes and 15 minutes at 30 Hz.The hypothesis of cellulose increasing recalcitrance was not supported as demonstrated in unambiguous carbon 1 to 4 of cellulose peaks for crystalline and amorphous cellulose.The 1500 ms CP-PDSD carbon 1 to carbon 6 transfers provide similar insight.The proportion of the crystalline to amorphous cellulose for the glucose carbon 1 to carbon 6 transfer appears to decrease more for the 2-minute milling period, to 90.47 ± 0.90% and 87.38 ± 0.88% respectively.The signal intensity for the crystalline cellulose and amorphous cellulose appear to be nearly equal for the glucose carbon 1 to carbon 6 after 15 minutes of milling.Both the carbon 4 and carbon 6 regions highlighted in Figure 7A–B appear to be low in signal intensity and it is worth noting the superposition of the noise over weak, broad peaks could distort the integrations so careful interpretation is necessary.Although the stems milled for 15 minutes show rigid cellulose within the fibril has greater amorphous cellulose than crystalline cellulose , the low signal intensity makes this observation somewhat ambiguous.The lower overall signal intensity of the stems milled for 15 minutes means the noise is superimposed over the tops of the peaks, compounding the error in these results.This factor is particularly relevant for the sample milled for 15 minutes given the signal intensity decreases by at least 80% for all peaks.For this purpose, over interpretation may be a liability when assessing recalcitrance using carbon 6 signals of cellulose in the 2D CP-PDSD experiments where cellulose carbon 4 chemical shift changes provide more information on recalcitrance due to morphology changes from crystalline to amorphous cellulose.Where the 1500 ms 2D CP-PDSD can give some insight into the larger cellulose structure, the 30 ms 2D CP-PDSD experiment reports on the D-glucose subunit of the cellulose polymer.Similarly, the 30 ms CP-PDSD experiment showed an overall decrease in cellulose structures was negligible after 2 minutes and >86% after 15 minutes of milling.When signal intensity is severely reduced the interpretation of integrations is less reliable due to noise super imposed over the tops of peaks.For this study, the 2DPDSD experiments, interpretations of carbon 1 to 4 peaks are more reliable than the carbon 1 to 6 signals.The proportion of the crystalline to amorphous cellulose for the glucose carbon 1 to carbon 4 transfer appears to change more for the 2-minute milling period to 101.53 ± 1.69% and 87.91 ± 1.00%, respectively.The signal intensity for the crystalline cellulose and amorphous cellulose appear to be nearly equal for the glucose carbon 1 to carbon 4 after 15 minutes of milling.