Research Article pubs.acs.org/journal/ascecg
Effect of in Vivo Deuteration on Structure of Switchgrass Lignin Xianzhi Meng,† Barbara R. Evans,‡ Chang Geun Yoo,§ Yunqiao Pu,§ Brian H. Davison,§ and Arthur J. Ragauskas*,†,§,∥ †
Department of Chemical & Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, Tennessee 37996, United States ‡ Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States § Biosciences Division, BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States ∥ Center for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University of Tennessee Knoxville, Institute of Agriculture, Knoxville, Tennessee 37996, United States S Supporting Information *
ABSTRACT: Biomass deuteration is an effective engineering method that can be used to provide key insights into understanding of biomass recalcitrance and the complex biomass conversion process. In this study, production of deuterated switchgrass was accomplished by growing the plants in 50% D2O under hydroponic conditions in a perfusion chamber. Cellulolytic enzyme lignin was isolated from deuterated switchgrass, characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) and compared with its protiated control sample to determine the effect of in vivo deuteration on the chemical structure of lignin. FTIR results showed that D2O can be taken up by the roots and transported to the leaves, and deuterium was subsequently incorporated into hydroxyl and alkyl groups in the plant and its lignin through photosynthesis. According to GPC results, deuterated lignin had slightly higher molecular weight, presumably due to isotope effects. 31P and heteronuclear single quantum coherence (HSQC) NMR results revealed that lignin in the deuterated biomass preserved its native physicochemical characteristics. The conserved characteristics of the deuterated lignin show its great potential applications for structural and dynamic studies of lignocellulose by techniques such as neutron scattering. KEYWORDS: Deuteration, Cellulolytic enzyme lignin, In vivo, Switchgrass, Neutron scattering
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INTRODUCTION
different biomass components such as cellulose, hemicellulose, and lignin within intact structures by phase contrast variation, enabling simultaneous observation of each component, particularly if one component has been enriched in D.9,12 Unlike isotopic labeling of synthetic polymers, biomass can directly incorporate labeled material from D2O in the environment via photosynthesis. Even when plants are grown in natural abundance water which has a concentration of D2O ranging from 0.0145 to 0.0149 mol %, the incorporation of D into plant tissues has been used to determine the differences in carbon fixation and transpiration between plants.13,14 As the D2O concentration increased, the unique properties of D2O such as higher viscosity begin to affect cell growth and metabolism. Thus, production of deuterated biomass is usually accomplished via deuterium enriched hydroponic cultures.12,15 Various deuterated biological materials from plants including switchgrass, winter grain rye, and annual rye grass have been
Growing concerns about environmental stewardship and diminishing availability of petroleum-based reserves have driven scientists to develop renewable biofuels from diverse sources such as lignocellulosic biomass.1 Biomass recalcitrance, the complex characteristics of lignocellulose to protect its carbohydrates from deconstruction by enzymes, remains the major barrier to a cost-efficient conversion of biomass to biofuels.2 The structure of lignocellulose plant cell walls contributes to biomass recalcitrance, and it has been characterized by a variety of analytical techniques.3−8 Smallangle neutron scattering (SANS) has been proposed and used for the characterization of lignocellulosic substrates as a nondestructive promising technique due to its unique sensitivity to hydrogen and ability to exchange thermal energies with the materials.9,10 Hydrogen (H) and deuterium (D) interact with neutrons quite differently, and substituting the hydrogen atoms with deuterium greatly enhances the potential of SANS due to the fact that contrast variation significantly depends on the differential scattering power of the D compared to H.11 Therefore, production of deuterated plant materials is crucial for SANS studies as it makes it possible to separate © 2017 American Chemical Society
Received: May 19, 2017 Revised: July 13, 2017 Published: July 27, 2017 8004
DOI: 10.1021/acssuschemeng.7b01527 ACS Sustainable Chem. Eng. 2017, 5, 8004−8010
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physiochemical characteristics of native lignin and thus can be used as a model in SANS experiments. However, to our current knowledge, there have been no studies on the effect of in vivo deuteration on lignin characteristics during the incorporation of D into the plant cell wall via growth in deuterium-enriched hydroponic cultures. Lignin represents one of the most important recalcitrance contributors in plants, thus it is essential to determine if the D substitution in plants can be achieved with minimal structural effects on lignin, which would support the use of neutron contrast variation methods for future lignin structural studies with SANS techniques. In this study, we investigated the effect of in vivo deuteration on the structure of lignin isolated from deuterated switchgrass. Partially deuterated and fully protiated plants were harvested from switchgrass grown hydroponically from tiller cutting in 50% D2O and 100% H2O, respectively. It was based on a previously published novel technique to produce partially deuterated switchgrass which is the first demonstration of a sufficiently deuterated perennial herbaceous feedstock to allow contrast matching SANS applications.17 Cellulolytic enzyme lignin (CEL) was isolated from these protiated and deuterated plants. Lignin samples were characterized for chemical structure, molecular weight, amount of various types of hydroxyl groups, and relative abundance of lignin subunits and common interlinkages by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), and 31P and heteronuclear single quantum coherence (HSQC) NMR. The findings will provide fundamental scientific insight critical to future SANS applications.
obtained by hydroponic cultivation using growth medium consisting of basal mineral salts in different levels of D2O.12,16,17 Besides deuterated plants, 13C labeled plants can also be achieved by continuous exposure of the plant to labeled CO2 with an isotopic composition that differs from ambient conditions during photosynthesis.18 Both 13C and D labeled lignin can be used to provide insights into the behavior of monolignol in lignin biosynthesis. With the position of the isotopic atoms in the product, the reaction pathway the initial metabolites utilize can be determined. For example, Chen et al. investigated the biosynthetic pathways to monolignols in Magnolia kobus by feeding stems with a D-labeled coniferyl alcohol, showing that the change from guaiacyl to syringyl units occurred at the cinnamyl alcohol stage.19 In addition, coniferin and syringin, deuterated at their methoxyl group, were also reported to be incorporated into lignin without major effect on plant growth and lignification.20 Deuterated synthetic lignin was fed to rats in a recent study, and the detection of deuterated mammalian lignans enterolactone clearly demonstrated for the first time that lignins are precursors of lignan phytoestrogens.21 Deuterated lignin model can be also used to provide insights into the mechanisms of reaction involving lignin. For example, the influences of deuteration of phenolic and nonphenolic lignin model dimers on the yields of the cleavage products in lignin pyrolysis were recently used to indicate the radical chain reactions pathways.22 13C-Labeled lignin is particularly useful for elucidation of lignin chemical structures in NMR application to avoid extensive peak overlaps resulting from the complex structure of lignin. On the other hand, the biggest advantage of D-labeled lignin as compared to 13 C labeled lignin is probably its unique application in neutron scattering experiments. The neutron scattering contrast among different lignocellulosic biomass components is normally small; however, this contrast in a neutron scattering experiment could be significantly enhanced by deuteration of a particular component such as lignin.10 The use of deuterated biomass in neutron scattering studies requires analysis of D incorporation in biomass and examination of its effects on the structure of cellulose, hemicellulose, and lignin. Previous studies reported that the replacement of H with D could be easily confirmed by FTIR and the level of D incorporation could be further quantified by a whole cell wall 1H and 2H liquid-state NMR.23 While there are still technical issues associated with production of deuterated materials that need to be addressed such as growth inhibition by D2O especially at the high levels of incorporation required by SANS, it is probably more important to determine if deuteration changes the physiochemical characteristics of native plant cell wall grown in H2O.24,25 Evans et al. studied the effect of D2O on growth properties and chemical structure of annual ryegrass (Lolium multiflorum) and reported that the annual ryegrass grown in 50% D2O was shorter and thicker in appearance than its protiated control but did not appear abnormal in leaf shape and coloration.12 More importantly, their study clearly indicated that deuterated ryegrass had slightly higher cellulose molecular weight compared to its protiated control, while hemicellulose molecular weights and cellulose crystallinity remain nearly unaffected with the deuteration.12 Similarly, a separate study also revealed the physicochemical properties of cellulose isolated from ∼34% deuterated switchgrass cultivated in 50% D2O basically resembled that of its protiated control.17 It is reasonable to hypothesize that deuterated lignin is able to preserve the major
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MATERIALS AND METHODS
Chemicals and Materials. Deuterium oxide (99.8%) was purchased from Cambridge Isotope Laboratories (Cambridge, Massachusetts). Plant jars, basal salt mixtures, agar, and growth hormones were all purchased from Phytotechnology Laboratories (Shawnee Mission, Kansas). The enzyme mixtures (Cellic CTec 2 and HTec 2) were provided by Novozymes (Franklinton, NC). Acetic anhydride, deuterated chloroform, chromium(III) acetylacetonate, deuterated chloroform, endo N-hydroxy-5-norbene-2,3-dicarboxylic acid imide, and 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane were purchased from Sigma-Aldrich (St. Louis, MO). Anhydride pyridine, ethanol, 1,4-dioxane, and toluene were obtained from VWR (Suwanee, GA). 1,4-Dioxane was distilled over NaBH4 prior to use, and other chemicals were all used as received. Switchgrass Cultivation. Switchgrass seeds (Panicum virgatum, Alamo cultivar) were obtained from the Bioenergy Science Center located at Oak Ridge National Laboratory, Oak Ridge, TN, USA. Perfusion chambers were assembled from 1 L graduated cylinders and perfused with dried ambient air supplied by an aquarium pump as described previously.17 Incident light intensity, diurnal cycle, and other plant growth conditions are shown in the Supporting Information. Switchgrass tiller were harvested at 1−2 month intervals due to height constraints of the growth chambers. Fourier Transform Infrared (FTIR). A Spectrum One FTIR system (PerkinElmer, Wellesley, MA) with a universal attenuated total reflection (ATR) accessory was used to characterize protiated and deuterated switchgrass and lignin. Each sample was pressed uniformly against the diamond surface using a spring-loaded anvil. FTIR spectra were obtained by averaging 64 scans from 4000 to 800 cm−1 at 4 cm−1 resolutions. Baseline and ATR corrections for penetration depth and frequency variations were carried out using the Spectrum One software. Cellulolytic Enzyme Lignin Isolation. Cellulolytic enzyme lignin was isolated from deuterated and protiated switchgrass according to literature procedure.26 Briefly, samples were milled and screened to a 0.42 mm using a Wiley mill (Thomas Scientific, Swedesboro, NJ), and 8005
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Figure 1. FTIR spectra of protiated and deuterated switchgrass (A) and lignin (B). with 256 data points, a 90° pulse, a 1.0 s recycle delay, a JC−H of 145 Hz, and 32 scans. Relative lignin interunit linkages abundance and monomer compositions were determined by using volume integration of contours in HSQC spectra semiquantitatively.27−29 NMR data and spectra processing was performed using TopSpin 2.1 software (Bruker BioSpin) and Adobe Illustrator CC (Adobe Inc.).
then Soxhlet-extracted with dichloromethane for 24 h. The extractivesfree samples were ball-milled in a porcelain jar with ceramic balls via Retsch PM 100 (Newton, PA) at 600 rpm for 2.5 h. The ground powder was then subjected to enzymatic hydrolysis in acetate buffer (pH 4.8, 50 °C) using CTec 2 and HTec 2 as the enzyme (150 mg protein loading/g biomass) for 48 h. The residue was then isolated and hydrolyzed one more time with freshly added enzyme and buffer. The recovered solids were then treated with protease (Sigma-Aldrich) at 37 °C overnight to remove any remaining enzymes, followed by a deactivation process done at 100 °C for 10 min. The freeze-dried lignin-enriched residue was finally extracted twice with 96% p-dioxane/ water mixture at room temperature for 48 h. The extracts were combined, rotary evaporated, and freeze-dried to get cellulolytic enzyme lignin. Lignin Molecular Weight Analysis. Lignin samples (∼5 mg) were acetylated using acetic anhydride/pyridine (1:1, v/v, 5 mL) at room temperature for 24 h. After 24 h, ethanol was added to the reaction and the mixture was then subjected to a rotary evaporator under reduced pressure. This process was further repeated until all the solvents were removed from the sample. The derivatized lignin was dissolved in tetrohydrofuran (THF) overnight prior GPC analysis. The molecular weight was analyzed by an Agilent GPC SECurity 1200 system equipped with several Waters Styragel columns (Waters Corporation, Milford, MA), an Agilent UV detector, and a refractive index detector. THF was used as the mobile phase (1.0 mL/min), and the injection volume was set at 30 μL. Data processing was performed by a Polymer Standard Service WinGPC Unity software, and the number-average and weight-average molecular weights were calculated based on a universal polystyrene calibration curve prepared by polystyrene standards. 31 P Nuclear Magnetic Resonance. Quantitative 31P NMR spectra were acquired on a Bruker Avance 400 MHz spectrometer equipped with a BBO probe using an inverse-gated decoupling pulse sequence (Waltz-16), 90° pulse, 25 s pulse delay with 128 scans. Lignin samples (∼20 mg) were dissolved in a solvent mixture of pyridine and deuterated chloroform (1.6/1.0, v/v, 0.50 mL). The lignin solution was then further derivatized with 0.075 mL 2-chloro4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). Chromium acetylacetonate and endo-N-hydroxy-5-norbornene-2,3-dicarboximide (NHND) were also added into the solution as relaxation agent and internal standard, respectively. All the data was processed using the TopSpin 2.1 software (Bruker BioSpin). Heteronuclear Single Quantum Coherence (HSQC) Nuclear Magnetic Resonance. Two-dimensional HSQC experiments were performed in a Bruker Avance 500 MHz spectrometer. A standard Bruker pulse sequence, hsqcetgpsp (phase-sensitive gradient-edited2D HSQC using adiabatic pulses for inversion and refocusing), was used under the following conditions: 11 ppm spectra width (5498 in Hz) in F2 (1H) dimension with 2048 data points (186.2 ms acquisition time) and 210 ppm spectral width in F1 (13C) dimension
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RESULTS AND DISCUSSION FTIR Characterization. FTIR was used to provide valuable chemical structural information on the deuterium incorporation into the plant and more specifically, lignin. All the samples showed strong FTIR bands at 2924 and 1249 cm−1, attributed to C−H stretching vibrations and C−H bending modes, respectively.30 The nonexchangeable deuterium substitution was confirmed by appearance of C−D stretching band in FTIR around 2165 cm−1 (Figure 1). FTIR clearly shows C−D bond in both biomass and the isolated lignin, and that the relative peak heights are comparable to those reported earlier for biomass and cellulose with D-incorporation quantified by NMR as 35−40%.17 The O−D stretching in deuterated switchgrass at ∼2495 cm−1 was attributed to the D−H exchange on the surface of biomass, while its absence in deuterated lignin sample indicated that the majority replacement of H by D probably happened at carbon−hydrogen sites instead of the hydroxyl groups of lignin (Figure 1B). However, this missing O−D stretching band in the FTIR of deuterated lignin could be also due to the fast hydrogen−deuterium exchange during the CEL isolation process. Besides the large band shifts observed for stretching bands of O−H and C−H compared to O−D and C−D, the isotope effect was also observed in other C−D deformation and vibration bands in deuterated lignin samples (Figure 1B). For example, the peak for C−H deformation at ∼1426 cm−1 showed a slight shift toward ∼1410 cm−1 in deuterated lignin. Similarly, the IR bands for aromatic ring stretching and vibrations occurred at ∼1598 and ∼1510 cm−1 also showed an isotopic shift in deuterated lignin toward smaller wavenumber around 1585 and 1491 cm−1, respectively. In conclusion, FTIR confirmed the nonexchangeable D substitution in both biomass and lignin samples, and the deuterated lignin sample has all the characteristic bands of typical native cellulolytic enzyme lignin, indicating that these two types of lignin appeared have qualitatively identical molecular structure. Lignin Molecular Weight Characterization. To determine the effect of deuteration on lignin structures during the 8006
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P NMR Characterization. Hydroxyl groups of lignin are another important characteristic possibly associated with lignin’s negative role in biomass recalcitrance. It has been proposed the content of phenolic and aliphatic hydroxyl groups in lignin could directly influence its hydrophobic interaction with enzymes which was identified as one of the major driving forces in the nonproductive adsorption of enzymes to lignin.10,31 31P NMR has been proved to be a fast and effective tool to determine contents of different types of hydroxyl group presented in lignin after phosphitylation.32 Quantitative information about various types of hydroxyl groups including aliphatic, carboxylic, guaiacyl, C5 substituted, and p-hydroxyphenyls are shown in Figure 4. The chemical shifts and
lignin biosynthesis process, GPC was used to measure the molecular weights of lignin isolated from the deuterated plant. Figures 2 and 3 illustrate the GPC chromatograms and
Figure 2. Molecular weight distribution curves of lignin isolated from deuterated and protiated switchgrass.
Figure 4. Hydroxyl group contents (mmol/g lignin) of lignin isolated from protiated and deuterated switchgrass as determined by 31P NMR.
integration regions for lignin in 31P NMR spectrum were summarized in Table 1. Aliphatic OH group appeared to be the Table 1. Typical Chemical Shifts and Integration Regions for Lignin in the 31P NMR Spectrum
Figure 3. Number-average (Mn) and weight-average (Mw) molecular weights of lignin isolated from protiated and deuterated switchgrass.
molecular weights of lignin isolated from switchgrass, respectively. Figure 2 indicates that the two types of lignin samples demonstrate a very similar shape of distribution curve. The quantitative molecular weight analysis showed that deuteration of switchgrass caused ∼20% and ∼19% increase in the weight-average (Mw) and number-average (Mn) molecular weight of lignin. The different molecular weight observed here is probably attributed to the substantial incorporation of D into the plant cell wall components including lignin. Previous studies with GPC analysis of carbohydrate also revealed a ∼ 22% and ∼34% increase of molecular weight of cellulose and hemicellulose after deuteration of switchgrass, which is consistent with other studies that characterized deuteration of bacterial cellulose and annual ryegrass.12,16,17 In terms of polydispersity index (PDI), unlike cellulose which increased its PDI from 3.8 to 4.9 after deuteration, lignin appeared unaffected in terms of PDI (2.23 vs 2.25) by deuteration.17 GPC results demonstrated that deuterated lignin had a slightly greater portion of higher molecular weight components due to the incorporation of D into lignin. The nearly identical PDI implied that in vivo deuteration in 50% D2O had a neglectable effect on the broadness of molecular weight distribution of lignin.
δ (ppm)
hydroxyl groups
133.6−136.0 ∼137.8 139.0−140.2 140.0−144.5 145.4−150.0
carboxylic acid OH p-hydroxyphenyl OH guaiacyl OH C5 substituted OH aliphatic OH
dominant hydroxyl groups, accounting for ∼77% and ∼78% of total free hydroxyl groups in protiated and deuterated lignin, respectively. Compared to protiated lignin, deuterated lignin appeared to have a 7.5% increase in aliphatic OH group. Among phenolic hydroxyl groups, p-hydroxyphenyl OH mostly attributed from the p-coumarate substructures was observed to the most prominent type with 0.44 mmol/g in protiated lignin and 0.55 mmol/g in deuterated lignin. However, the total amount of phenolic OH groups in deuterated lignin appeared to be comparable with that in the control sample. Both lignin samples had a trace amount of carboxylic OH groups. The 31P NMR results showed that the phenolic hydroxyl groups composition in lignin remained relatively stable during deuteration of the plant, and the slightly increase of aliphatic OH group in D-labeled lignin might possibly due to isotopic differences in intracellular transport during lignification and cell wall development process. 8007
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Figure 5. Aromatic regions of HSQC spectra of lignin isolated from protiated and deuterated switchgrass.
Figure 6. Aliphatic regions of HSQC spectra of lignin isolated from protiated and deuterated switchgrass.
HSQC NMR Characterization. Semiquantitative analysis of HSQC has been widely used to provide detailed information about the chemical structures of lignin, including monolignol compositions and relative abundance of interunit linkages.27,33,34 Figures 5 and 6 presented the aromatic and aliphatic regions of HSQC NMR spectra of lignin isolated from protiated and deuterated switchgrass. The 13C/1H cross-peaks in these regions were assigned according to literature and presented in Table 2. Both protiated and deuterated lignin appeared primarily composed of syringyl (S) and guaiacyl (G)
units along with considerable amounts of p-coumarate (Figure 5). It was found that protiated and deuterated lignin demonstrated similar structural features. S units showed major cross peaks for the C2,6/H2,6 correlations centered at 103.5/6.69 ppm, whereas the G unit shows correlations for C2/ H2, C5/H5, and C6/H6 around 111.0/6.97, 114.9/6.79, and 118.9/6.82 ppm, respectively. The p-hydroxyphenyl (H) unit was also readily identified due to the presence of its diagnostic cross peaks around 127.4/7.18 ppm (H2/6). In addition, the spectra also contained a considerable amount of p-coumarate 8008
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ACS Sustainable Chemistry & Engineering Table 2. Signal Assignments of Chemical Structures in 13 C−1H HSQC NMR spectra18 δC/δH (ppm)
assignment
54.1/3.44 55.6/3.73 59.8/3.63 71.1/4.18 71.1/4.73 71.4/4.85 83.7/4.31 84.9/4.66 84.6/4.71 86.7/5.45 94.4/6.55 99.0/6.22 103.5/6.69 104.1/7.31 111.0/6.97 111.1/7.30 114.9/6.79 118.9/6.82 127.4/7.18 129.5/7.41 144.5/7.47
Cβ/Hβ in phenylcoumaran (β-5) C/H in methoxyl group Cγ/Hγ in β-aryl ether (β-O-4) Cγ/Hγ in resinol (β−β) Cα/Hα in β-O-4 linked to a G unit Cα/Hα in β-O-4 linked to a S unit Cβ/Hβ in β-O-4 linked to a G unit Cβ/Hβ in β-O-4 linked to a S unit Cα/Hα in resinol (β−β) Cα/Hα in phenylcoumaran (β-5) T8 in tricin (T) T6 in tricin (T) C2,6/H2,6 in syringyl (S) unit T2′, T6′ in tricin C2/H2 in guaiacyl (G) unit C2/H2 in ferulate (FA) C5/H5 in guaiacyl (G) unit C6/H6 in guaiacyl (G) unit C2,6/H2,6 in p-hydroxyphenyl (H) unit C2,6/H2,6 in p-coumarate (pCA) Cα/Hα in pCA/FA
Table 3. Semiquantitative Information for Lignin Subunits and Interunit Linkages in Lignin Isolated from Protiated and Deuterated Switchgrassa lignin chemical structures
protiated switchgrass
deuterated switchgrass
Lignin Subunits 42.4 54.8 2.8 0.77 Hydroxycinnamates ferulate (FA) 12.2 p-coumarate (p-CA) 38.0 Lignan tricin (T) 2.1 Lignin Interunit Linkages β-aryl ether (β-O-4) 26.2 resinols (β−β) 0.5 phenylcoumaran (β-5) 5.4 syringyl (S) guaiacyl (G) p-hydroxyphenyl (H) S/G ratio
a
43.0 55.1 1.9 0.78 12.3 38.4 2.3 28.3 0.6 6.2
Content (%) expressed as a traction of S + G + H.
lignin had slightly higher molecular weight than protiated lignin. No significant differences were obtained in lignin structure between protiated and deuterated sample. Thus, lignin characterization results obtained here support the development and utility of deuterated plant/lignin to characterize biomass through neutron scattering studies in the future.
(pCA) with a strong cross peak centered at 129.5/7.41 ppm and correlations peaks overlapping with G5 around 114.9/6.79 ppm representing the C2,6/H2,6 and C3,5/H3,5 of pCA, respectively. Ferulate (FA) signals were observed with cross peaks around 111.1/7.30 ppm (FA2). Signals associated with vinyl carbons in C7 arose from pCA and FA were observed with cross peaks around 144.5/7.47 ppm. It has been reported that tricin, a flavonoid type compound, can be covalently incorporated into lignin in corn stover, rice straw, sugar cane, and wheat straw.33−36 Herein, the correlations for C2′/H2′ and C6′/H6′ of tricin were observed in aromatic regions at 104.1/ 7.31 ppm (Figure 5). Two other strong and well-resolved C/H signals at the 6 and 8 position of tricin were also readily observed at 99.0/6.22 and 94.4/6.55 ppm, respectively. In aliphatic region of lignin (Figure 6), signals associated with methoxyl (55.6/3.73 ppm) and β-O-4 were among the most prominent ones. The cross peaks at 71.4/4.85 ppm and 86.7/5.45 ppm were ascribed to the correlation of α position of β-aryl ether (β-O-4) and phenylcoumaran (β-5), respectively. Resinol substructure (β−β) was barely detected around the noise level in lignin isolated from switchgrass grown in H2O, while it was missed in deuterated switchgrass. The contents of lignin subunits, hydroxycinnamates, and relative abundance of interunit linkages are shown in Table 3. Protiated lignin had slightly higher content of H unit compared to deuterated lignin. On the other hand, contents of β-O-4, β-5, and β−β were slightly higher in deuterated lignin. In conclusion, no significant effects of deuteration on the lignin structure were observed.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01527. Plant cultivation section (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Fax: 865-974-7076. Tel.: 865-974-2042. E-mail: argauskas@ utk.edu (A.J.R.). ORCID
Barbara R. Evans: 0000-0002-2574-2567 Arthur J. Ragauskas: 0000-0002-3536-554X Funding
This research is funded by U.S. Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research, under the Genomic Science Program (FWP ERKP752). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in according with the DOE Public Access Plan (https://www.energy.gov/
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CONCLUSIONS Plant deuteration is a key step for realizing the full potential of neutron scattering studies. Deuterium was incorporated into the lignin through a long-term continuous production of switchgrass grown hydroponically in 50% D2O. For the first time, in vivo deuterated lignin isolated from plant without addition of deuterium-labeled lignin precursors was characterized in detail by various analytical techniques. Deuterated 8009
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ACS Sustainable Chemistry & Engineering
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downloads/doe-public-access-plan). We would like to thank Dr. Clemens Anklin from Bruker BioSpin for helping us run NMR experiments.
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DOI: 10.1021/acssuschemeng.7b01527 ACS Sustainable Chem. Eng. 2017, 5, 8004−8010
