Article pubs.acs.org/JAFC
Effect of D2O on Growth Properties and Chemical Structure of Annual Ryegrass (Lolium multif lorum) Barbara R. Evans,† Garima Bali,‡ David T. Reeves,†,∥ Hugh M. O’Neill,§ Qining Sun,‡ Riddhi Shah,§ and Arthur J. Ragauskas*,‡ †
Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831, United States Institute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0620, United States § Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States ‡
ABSTRACT: The development of deuterated biomass is essential for effective neutron scattering studies on biomass, which can provide key insights into the complex biomass conversion processes. A method for optimized production of deuterated annual ryegrass (Lolium multif lorum) was developed by growing the plants in 50% D2O in perfused hydroponic chambers. Deuterium incorporation of 36.9% was found in the annual rye grown in 50% D2O. Further, deuterium incorporation of 60% was achieved by germinating the rye seedlings in H2O and growing in 50% D2O inside the perfusion chambers. The characteristics related to enzymatic hydrolysis such as biomass composition, degree of polymerization, and cellulose crystallinity were compared with its control protiated counterpart. The cellulose molecular weight indicated slight variation while hemicellulose molecular weights and cellulose crystallinity remain unaffected with the deuteration. KEYWORDS: Lolium multif lorum, hydroponics, crystallinity, 13C CPMAS, deuteration
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Since the discovery of deuterium (2H) and the development of methods to purify deuterium oxide (D2O), it has been employed as a uniquely useful tool for biological studies. Deuterated biological materials as well as incorporation of deuterium from D2O by living organisms have proven useful for proton magnetic resonance (1H NMR), isotopic replacement to facilitate interpretation of reaction pathways, and metabolic analyses6,7 including nutritional studies in humans.8,9 Plants are normally exposed to low levels of D2O in the environment. The concentration of D2O in fresh water has been reported to range from 0.0145 to 0.0149 mol percent.6 Even at these low concentrations, incorporation of deuterium into plant tissues, particularly cellulose, has been used to examine differences in carbon fixation and transpiration between plants.10 As the concentration of D2O is increased, the physical and chemical differences between D2O and H2O begin to affect the metabolism and growth of living organisms. D2O has higher viscosity, a higher melting point, and a higher boiling point than H2O. Salts and gases have lower solubility in D2O. Ionization constants are one-fifth that of water, and amino acids show a 0.5 pK unit increase in their apparent ionization constants. This directly impacts availability of CO2 for photosynthesis, as it reduces both dissolution of CO2 and formation of carbonic acid, as well as ionization of carbonic acid to bicarbonate. The kinetic isotope effects are due to the difference in chemical bond reactivity for bonds to D compared to bonds to H. In
INTRODUCTION The conversion of lignocellulosics into bioethanol remains a challenging process due to the complex cell wall structure and the recalcitrant nature of biomass.1 A multilength structural characterization of such biomass is essential to better understand the recalcitrance and the deconstruction mechanism during the biological and thermochemical conversion of various potential energy crops. Small angle neutron scattering (SANS) provides a useful tool for investigation of lignocellulosic structural complexity and its changes during pretreatment and hydrolysis to sugars.2 Its unique advantages for structural analysis of biological materials arise mainly from the strong interactions of neutrons with the hydrogen or its deuterium isotope. However, deuteration of the biomass samples is vital to realize the full potential of this technique as contrast variation greatly depends on the differential scattering power of deuterium compared to hydrogen; therefore, substituting the protons with deuterium significantly enhances the signal-tonoise ratio of the deuterated component.3−5 Thus, the reduction of hydrogen background scattering requires deuterium enrichment at higher levels than typical isotopic tracer studies to achieve the high signal-to-noise ratio. Particularly valuable are experiments that examine scattering length density variation between elements and isotopes. By varying the isotopic content of the solvent water, the contrast between the scattering by the solute and that from the solvent is changed to enable extraction of shape information from the scattering patterns. Similarly, use of deuterated plant materials can greatly increase the value of SANS studies, as the scattering patterns of the different biomass components can be separated by phase contrast, enabling simultaneous observations of each component. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2595
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cobalt chloride hexahdrate, 0.2 cupric sulfate penthydrate, 20 mg/L Na2EDTA dihydrate, 15 ferrous sulfate heptahydrate, 195.4 mg/L anhydrous magnesium sulfate, 10 mg/L managanese sulfate monohydrate, 0.1 mg/L sodium molybdate dehydrate, 1 mg/L potassium iodide, 2500 mg/L potassium nitrate, 1 mg/L zinc sulfate heptahydrate, pH 4.2. Before seeding, the seeds were surface-sterilized with 70% ethanol for 2 min and then soaked in Schenk and Hildebrandt’s basal salts for plants (S&H salts) in H2O for 15 min. Initial determination of optimal D2O concentration was established by germination and growth of seeds in a concentration range of 0 to 99.8% D2O in jars for 26 days. The Mettler AE200 analytical balance used for weighing the deuterated plants and for weighing reagents has an accuracy of ±0.0001 g. The Ohaus CentOGram triple beam balance that was used to weigh the control ryegrass has an accuracy of ±0.01 g. Concentration Dependence of Deuteration. The effect of D2O concentration on growth of annual ryegrass and deuterium incorporation in biomass was initially determined using sealed glass jars with 3-mm glass beads as supports. Growth solutions were Schenk and Hildebrandt’s salts (Phytotechnology Laboratories, Shawnee Mission, KS) prepared in 0, 20, 40, 60, 80, and 99.8% D2O. Ten seeds were used per jar. Germination was scored at 14 days and plants were harvested at 26 days for analysis. Hydroponic Cultivation in Perfusion Chambers. Deuterium enrichment of plants requires enclosure and perfusion with dried air to limit exchange with ambient water vapor as well as to limit D2O usage and avoid microbial contamination. Since it was desirable to obtain multiple biomass samples grown at different concentrations of D2O, a system that allows simultaneous cultivation of several plants in individual isolated chambers was devised. Suitable chambers for longterm cultivation of grasses in D2O mixtures were constructed from graduated glass cylinders (Figure 1). Standard 1 and 3 L graduated glass cylinders were modified by the in-house glass shop at ORNL by cutting off the top 2 cm to remove the pouring lips followed by glass polishing to enable a tight fit with the rubber stoppers used as closures. The 1 and 3 L chambers were fitted with size 12 two-hole rubber stopper and three-hole size rubber stoppers respectively. Two 20.0 cm
living organisms, this resulted in a lag time in growth, which can be overcome experimentally for some species such as algae and bacteria by adaptation to increasing amounts of D2O.6,7 Higher plants have been reported to tolerate 30−70% D2O, with adverse effects increasing with D2O concentration. Metabolic responses of plants to higher concentrations of D2O vary among species. Abnormal morphology and delayed development are noted at concentrations of D2O above 50% for many species.7,11 Seed germination and root elongation are adversely affected as D2O concentrations are increased with wheat,12 rye,13 and Arabidopsis.11 To avoid the problems observed at 50% and higher concentrations of D2O, plants can be cultivated at lower concentrations if sufficient deuterium incorporation can be attained for the intended use such as characterization using neutron scattering studies. Partially deuterated carrots, kale, and spinach were grown hydroponically in solutions of 15−30% D2O to obtain isotopically labeled vegetables for nutritional studies.8,9 Despite numerous studies being performed on the effect of deuteration on plant metabolic pathways, the impact of deuteration on individual biomass components for neutron scattering studies has not so far been investigated. In such a scenario, it is important to corroborate the methods for production of deuterated biomass and the chemical characterization of partially deuterated biomass components for progress of biofuel through neutron scattering technologies. Annual or Italian ryegrass, Lolium multif lorum, is a C3 grass widely used across the world as a forage and cover crop. In the southeastern United States, the land area planted in L. multif lorum has increased from 1.1 million ha in 200514 to 1.3 million ha in 2010.15 Due to its importance in agriculture, the composition and genetics of annual ryegrass have been extensively studied. In addition to its widespread agricultural use, Lolium ryegrass is frequently used for both field and laboratory research to investigate endophytes16 phytoremediation,17 pollutant uptake and toxicity,18−22 morphological responses,23 herbicide resistance24 and studies of plasma membranes and associated enzymes such as glucan synthases.25,26 In this study, we investigated the effects of D2O and high levels of deuterium enrichment on the growth and morphology of annual ryegrass (Lolium multif lorum), an important and intensively studied forage and cover crop, for future structural studies with SANS applications. The laboratory grown deuterium-labeled annual rye was analyzed for its deuterium incorporation levels by an NMR method.
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MATERIALS AND METHODS
Substrate. Annual rye (Lolium multif lorum) seeds were obtained from a local gardening supply store. Identification of the species as Lolium multif lorum, an amenity and forage grass commonly cultivated in the southeastern United States, was confirmed based on the following characteristics exhibited by plants grown both in open pots in soil and in enclosed hydroponic conditions: plant height 70−80 cm at 60−100 days growth; absence of tillering; inflorescence head formation after 60 days; influorescence stems bare at base; shape of spikelets on influorescence stems; and presence of awns on seeds. The growth medium used for plant cultivation was Schenk and Hildebrandt’s basal salts (Phytotechnology Laboratories, Shawnee Mission, KN) dissolved in Milli-Q (Millipore) water. The composition of the Schenk and Hildebrandt’s basal salts for plants when prepared at 3.2 g/L in distilled water is given by the manufacturer (Phytotechnology Laboratories) as: 300 mg/L ammonium phosphate monobasic, 5 mg/L boric acid, 151 mg/L anhydrous calcium chloride, 0.1 mg/L
Figure 1. Assembled growth chamber with the labeled component parts. 2596
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lengths of 0.6 cm diameter glass tubing with 90-degree bends at 7.6 cm were inserted into the stoppers. Heat-resistant silicone rubber tubing was used to connect the glass tubing to sterile 0.22 μm pore size, 33 mm diameter syringe filters (Millipore Ireland Ltd., Tullagreen, Carrigtwohill, Cork, Ireland) and to connect the tubing to PE drying tubes containing Eagle silica gel desiccant with color indicator. Glass Y-connectors were used to connect multiple chambers using the silicone tubing. Plants were perfused with ambient air from the growth room using Marina 50 and Marina 75 aquarium pumps (Rolf Hagen (USA) Corp., Mansfield, MA) that were connected to the chambers by sterile syringe filters connected to Elite aquarium tubing, with in-line valves for flow rate adjustment. The CO2 concentration of the ambient air filtered through the silica gel desiccant ranged from 385 to 465 ppm as determined by measurement with an LI-6252 CO2 analyzer (LICOR, Lincoln, NE) that was calibrated with analyzed compressed air mixtures (Air Liquide, Oak Ridge, TN). Airflow into each chamber was maintained at 50−100 mL/min as measured with an Alltech Digital Flowcheck HR meter. Due to the small volumes of growth medium used per chamber and to limit evaporation losses, the chambers were perfused from the top and the growth solutions were not aerated. Two types of growth lamps were used: a metal halide lamp mounted on a stand and fluorescent Sylvania Gro-Lamps on a plant cart (Carolina Biological Supply Company, Burlington, NC). The metal halide lamp was mounted in a SunSystem 2 lamp holder (Future Garden, North Lindenhurst, NY) that was suspended from an in-house built lamp stand. Average incident light intensity was 150 μmol photons/m2/s as measured with a LICOR-250 Light Meter equipped with a quantum sensor (LI-COR, Lincoln, NE) and 8930 lx as measured with a Traceable Light Meter (International LLC, Radnor, PA). Diurnal cycles were 12 h light, 12 h dark. Growth medium was Schenk and Hildebrandt’s basal salts (Phytotechnology Laboratories, Shawnee Mission, KN) dissolved in Milli-Q (Millipore) water. Deuterium oxide (99.8%) was purchased from Cambridge Isotope Laboratories (Cambridge, MS). Hydroponic baskets with 5 cm diameter were used to support the rye seeds on 47-mm analytical glass fiber filters during growth and germination. Ambient temperature in the plant growth area was 27 °C during illumination and 21 °C during the diurnal dark period. Cultivation of Annual Ryegrass in 50% D2O for Production of Deuterated Biomass. Initial determination of optimal D2O concentration was established by germination and growth of seeds in a concentration range of 0−99.8% D2O in jars for 26 days. Deuterium incorporation did not increase above 60% D2O, while seed germination decreased above 60% (Figure 2). Based on the preliminary tests, a concentration of 50% D2O was used for scale-up of biomass production in the perfusion chambers. Seeds were germinated and seedling primary roots were established before transfer to the perfusion chambers. The treated seeds (thirty per dish) were germinated in 6-cm diameter polystyrene culture dishes on 47-mm glass filter disks containing 5% acrylic binder (Gelman Sciences/Pall Life Sciences, Ann Arbor, MI) moistened with 4 mL of S&H salts in H2O. The seeded dishes were incubated in the dark for 24 h, followed by illumination with a metal halide growth lamp on a 12 h diurnal cycle. Dark temperature range was 21 to 22 °C, and light temperature range was 25 to 28 °C. The seeds began germinating at 5 days. The lids were removed from the culture dishes and the dishes holding the seedling plants were transferred to 1 L glass plant jars 6−7 days after seeding. Another 2 mL of S&H salts were added to each dish after transfer to the jars. At eleven days after seeding, the glass filters holding the seedling plant samples #1, #2, and #3 were carefully placed in 5 cm hydroponic plant baskets. Each basket was placed into a 1L perfusion chamber and 12 mL of S&H salts in 50% D2O, 50% H2O were added to each cylinder. These plants were watered with S&H salts in 50% D2O, 50% H2O once or twice weekly by injection through the perfusion tubing using sterile syringes and syringe filters to ensure that the roots remained covered with solution. Plants appeared to grow normally, with the H2O-grown rye growing taller and faster than the plants growing in 50% D2O as expected, yielding several grams of biomass at harvest (Figure 3 and Table 1).
Figure 2. Germination and deuterium incorporation determined for annual ryegrass grown in increasing concentrations of D2O to establish the optimal cultivation conditions for deuterated biomass production.
Figure 3. Annual ryegrass plants grown in (A) 50% D2O under continuous air perfusion and (B) H2O-grown controls. The perfusion chamber design reduced evaporation and enabled cultivation for two months with cumulative usage of less than 100 mL solution per chamber. The total amount of hydroponic solution used for the cultivation of the protiated plants was 82 mL per plant, while the total amount of 50% D2O solution used for the deuterated plants was 88 mL/plant. The total amount of D2O used per plant for 50 days of growth in the 50% D2O solution was 44.3 ± 17.5 mL. The seedling 2597
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(10 mg) was then dissolved in a pyridinium chloride−DMSO solvent mixture as described previously.28 The NMR spectra of homogeneous biomass solution were recorded on a Bruker Avance 400 MHz spectrometer operated at 61.4 MHz for deuterium and 400.1 MHz for proton. 1H NMR spectra were measured at 55 °C with 64 k data points, a 10 s recycle delay, and 16 scans. Deuterium (2H) NMR spectra was recorded at 55 °C with 32 k data points, 10 s recycle delay, and 4000 scans. Carbohydrates and Klason Lignin Analysis. Samples for carbohydrate and acid-insoluble lignin analysis were prepared using a two-stage acid hydrolysis protocol based on Tappi method T-222 om88. The sugar solution was analyzed for carbohydrate components by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using Dionex ICS-3000 (Dionex Corp., USA).29 The standard deviation associated with lignin and carbohydrate analysis was 0.3 to 0.6 and 0.02 to 0.9, respectively. Cellulose and Hemicellulose Isolation. The extractive-free annual ryegrass samples were delignified using peracetic acid as described previously.30 Cellulose was isolated from the delignified sample (0.10 g) by extraction with a 17.5% NaOH solution (5.00 mL) at 25 °C for 2 h. The mixture was diluted to 8.75% NaOH solution by addition of 5 mL of deionized water and repeated stirring at 25 °C for an additional 2 h. The isolated α-cellulose samples were then collected by centrifugation, washed with an excess of deionized water and airdried. For hemicellulose isolation, the filtrate obtained above, was poured into a mixture of ethanol: acetic acid (70:30, v:v). The supernatant was removed by centrifugation and the precipitated hemicellulose was again washed with a mixture of ethanol: acetic acid (70:30, v:v) and air-dried for analysis. GPC Analysis of Cellulose. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) were determined by GPC after tricarbanilation of cellulose as described previously.31 The derivatized cellulose was dissolved in tetrahydrofuran (1 mg/mL), filtered through a 0.45 mm filter and placed in a 2-mL autosampler vial. The molecular weight distributions of the cellulose tricarbanilate samples were analyzed on an Agilent GPC Security 1200 system equipped with four Waters Styragel columns (HR1, HR2, HR4, and HR5), Agilent refractive index (RI) detector and Agilent UV detector (270 nm) using THF as the mobile phase (1 mL/min) with injection volumes of 0.02 mL. A calibration curve was constructed based on eight narrow polystyrene standards ranging in molecular weight from 1.5 × 103 to 3.6 × 106 g/mol. Data collection and processing were performed using Polymer Standards Service WinGPC Unity software (Build 6807). Molecular weights (Mn and Mw) were calculated by the software relative to the universal polystyrene calibration curve. Polydispersity index (PDI) was calculated by dividing Mw by Mn. GPC Analysis of Hemicellulose. The GPC analyses of hemicellulose samples were carried out using an Agilent 1200 series HPLC system consisting of an online degasser and an autosampler, a RI detector and three columns of Ultrahydrogel 120, 250, and 500 (Waters Inc., USA) linked in series. The RI detector was set at 35 °C. The mobile phase was an alkaline sodium hydroxide/acetate solution (0.20 M sodium hydroxide, 0.1 M sodium acetate, pH 12−13) and the flow rate was 0.5 mL/min. The hemicellulose samples were dissolved in mobile phase (∼1 mg/mL) and the solution was then filtered with a 0.2 μm filter. The filtered sample (25 μL) was injected into the GPC column system for analysis. Pullulan standard samples were used for the calibration.32 Sample Preparation for Solid-State Nuclear Magnetic Resonance for Crystallinity Measurement. The cellulose samples for NMR were prepared from the control protiated and deuterated annual ryegrass from the holocellulose sample (1.00 g) by mild acid 2.5 M HCl hydrolysis as described previously.33 The isolated αcellulose samples were then collected by filtration rinsed with an excess of deionized water and dried in a fume hood. For NMR analysis, 4-mm cylindrical ceramic MAS rotors were filled with the isolated α-cellulose. Solid-state NMR measurements were carried out on a Bruker Avance400 spectrometer operating at a frequency of 100.55 MHz for 13C in a Bruker double-resonance MAS probe at spinning speeds of 10 kHz.
Table 1. Net Biomass Yields from Annual Ryegrass Plants Grown Hydroponically with Perfusion (30 Seeds/Basket) plant stems
sample
growth solution
#1
start in H2O, then at 11 days start in 50% D2O start in H2O, then at 11 days start in 50% D2O start in H2O, then at 11 days start in 50% D2O H2O H2O H2O
#2 #3 #4 #5 #6
wet weight total (g) 9
3.55
15
6.18
13
6.35
4 6 4
1.18 1.45 2.59
samples #4, #5, and #6 were transferred to hydroponic baskets on the twelfth day after seeding, and all three baskets were placed in a 3L perfusion chamber to which 150 mL of S&H salts in H2O were added. Growth rates of the plants grown in H2O was observed to double following transfer to the perfusion chambers, while the growth rates of the plants transferred to 50% D2O in perfusion chambers did not change significantly compared to the initial growth rate in H2O before perfusion (Tables 2 and 3). At harvest, plants in their baskets were
Table 2. Growth Rates of Annual Ryegrass Plants Comparing Effects of 50% D2O and Perfusion Determined by Averaging the Slopes of the Linear Regression Lines from Tracking Growth of Seedlings as Shown in Figure 4a before perfusion sample sets 50% D2O H2O controls
growth rate (cm d−1) 0.399 ± 0.103 (ns) 0.396 ± 0.114
during perfusion
growth solution S&H in H2O S&H in H2O
growth rate (cm d−1)
growth solution
0.381 ± 0.065*
S&H in 50% D2O S&H in H2O
0.907 ± 0.096
a Errors are reported in term of standard deviation; ns = not significant, * = P ≤ 0.05 calculated by student t test.
Table 3. Plant Growth and Biomass Production was Averaged for the Three Sets of Protiated Plants and the Three Sets of Deuterated Plantsa growth solution
growth time (days)
tallest height at harvest (cm)
total biomass yield (mg stem−1 day−1)
50% D2O H2O
61 47
33.0 ± 4.5 (ns) 50.3 ± 8.8
7.07 8.40
a
Errors are reported in term of standard deviation; ns=not significant.
removed from the chambers, excess hydroponic solution was allowed to drain from the plants in a tray and then the roots and glass fiber supports were blotted with several Kim-Wipe tissues. The protiated control plants (#4, #5, and #6) were harvested at 47 days from seeding and stored at −20 °C until characterization and further use. The plants grown in 50% D2O (#1, #2, and #3) were harvested at 61 days and used fresh. Plant growth and biomass production of protiated plants and deuterated plants after 47 and 61 days respectively are presented in Tables 1, 2, and 3. Preparation of NMR Solution for Deuterium Incorporation Studies. Dimethyl sulfoxide (anhydrous, 99.9%), pyridinium chloride (98%), and TFA (reagent grade, 99.0%) were purchased from Sigma Aldrich. Dimethyl sulfoxide (DMSO)-d6 (99.9% D) and trifluoroacetic acid (TFA)-d (99.5% D) were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MS). Deuterated pyridinium chloride was synthesized according to a literature procedure.27 Ionic liquid solvent mixture was prepared by mixing vacuum-dried protio pyridinium chloride or deuterated pyridinium chloride with DMSO or DMSO-d6, respectively in 1:4 ratios in an inert atmosphere. Deuterated ryegrass 2598
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CP/MAS experiments utilizing a 5 ms (90°) proton pulse, 1.5 ms contact pulse, 4 s recycle delay and 4−8 K scans. All spectra were recorded on equilibrated moisture samples (∼35% water content). FTIR Spectroscopy. Annual ryegrass samples were vacuum-dried for 24 h. Infrared (FTIR) spectra were obtained using a Perkin-Elmer 100 Series Fourier transform infrared spectrometer in an attenuated total reflectance (ATR) mode. The spectra were recorded with the accumulation of 128 scans, a resolution of 4 cm−1 in the range from 4000 to 500 cm−1. After the baseline correction, the two spectra were scaled with respect to one another, using the C−O stretching of the glucopyranose ring at ∼1053 cm−1. Scanning Electron Microscopy (SEM). Scanning electron microscope images of solvent extracted and holopulps from protiated and deuterated annual rye samples were obtained with a LEO 1530 TFE-SEM microscope operated at 5 kV. The samples were freezedried and fixed on carriers with conductive carbon tape. The samples were coated with gold using an ion sputter coater and observed with a thermally assisted field emission (TFE) scanning electron microscope. Statistical Analysis. Student t tests were performed to determine significance between experimental values, where p ≤ 0.05 is 95% significance (*), and p ≤ 0.01 is 99% significance (**).
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RESULTS AND DISCUSSION Annual Ryegrass Cultivation in D2O. From previously reported studies,34−37 it was apparent that the effects of D2O on higher plant growth were dependent on species, concentration, and growth stage. The typically observed concentration dependence found little effect at concentrations up to 0.5%, delayed germination and slower growth at 50%, and complete inhibition, or very stunted and abnormal, germination and growth at concentrations higher than 80%. 38 As a result, deuterium enrichment of plants is usually carried out at concentrations less than 50% D2O using seedlings that have been sprouted and established in H2O.9,39 The annual ryegrass (Lolium multif lorum) for production of deuterated biomass were grown in enclosed chambers continually perfused with dry air to limit exchange with ambient water vapor while providing a constant supply of CO2 for photosynthesis and O2 for photorespiration. Initial screening found that plant germination was severely inhibited in 80% D 2 O while deuterium incorporation was not substantially improved (Figure 2). Annual ryegrass germinated in 70% D2O, but with very low germination rates and a growth rate of 21% of H2O-grown controls. Germination reached control levels in 50% D2O, but subsequent growth rates were 45% of H2O-grown controls (Figure 4). Vernalization, cold treatment of seeds, has been reported to improve growth of winter rye and brome grass at low temperatures.40 To examine whether vernalization would improve growth under deuterating conditions, seeds were incubated for 24 h at 4 °C followed by 10 days at 10 °C. Germination increased 4-fold in 70% D2O following vernalization, but growth rate and low biomass yield (less than 10% of controls grown in H2O) were not improved, so this approach was not further investigated. Growth rates of the plants grown in H2O were observed to double following transfer to the perfusion chambers, while the growth rates of the plants transferred to 50% D2O in perfusion chambers did not change significantly compared to the initial growth rate in H2O before perfusion (Figure 4). The annual ryegrass grown in 50% D2O was shorter and thicker in appearance than matched protiated controls, but did not appear abnormal in coloration or leaf shape (Figure 3). Possible internal changes in leaf and stem vascularization were not examined. The differences in morphology may correspond to the normal range of adaptations of Lolium ryegrass species and related fescue
Figure 4. Growth rates determined by linear regression of individual plant heights of (A) three sets of H2O control seedlings and (B) three sets of seedlings transferred to 50% D2O.
(Festuca) grasses to drought, cold, and light conditions,15,23 but this will need to be confirmed by comparative studies. NMR Analysis. The deuterium incorporation in annual ryegrass samples was examined using the methodology as described previously.28,41 NMR analysis of annual ryegrass samples grown in increasing D2O concentrations showed that the deuterium incorporation increased linearly with increasing D2O concentration up to 60% D2O, but was not improved significantly for annual rye grown in 80% D2O, while germination was also inhibited at 80% (Figure 2). Ultimately, based on the effect of D2O concentration on growth and biomass deuterium incorporation in the preliminary trials, annual rye grown in 50% D2O was selected for detail structural analysis. 1H and 2H NMR experiments were performed in order to determine the deuterium incorporation level in rye grown in 50% D2O. A standard inversion recovery pulse sequence was used to measure the longitudinal relaxation time for all the resonances and to determine the appropriate recycle delay. Figure 5 illustrates the 1H and 2H NMR spectra of annual ryegrass grown in 50% D2O. The 2H NMR spectrum of deuterated annual ryegrass shows the poorly resolved group of signals from 3.0 to 5.5 ppm, which is attributable to the cellulosic and other polysaccharides protons in the biomass. The protons from lignin side chain are also present in the described chemical shift range. The signals from 0.5 to 2.0 ppm are due to presence of side chain acetates and some residual 2599
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Figure 5. (A) 2H NMR and (B) 1H NMR spectra of deuterated annual ryegrass grown in 50% D2O; (C) 2H NMR and (D) 1H NMR spectra of control annual ryegrass. The region between two dotted lines represents protons/deuterium mostly from cellulose and some lignin side chains.
Table 4. Compositional Analysis of Annual Ryegrass (Based on Dried Biomass)a deuterated ryegrass protiated ryegrass a
Klason lignin (%)
arabinose (%)
galactose (%)
glucose (%)
xylose (%)
mannose (%)
10.2 ± 0.6* 12.8 ± 0.3
5.6 ± 0.02* 6.4 ± 0.02
3.8 ± 0.03* 4.4 ± 0.04
66.7 ± 0.9 58.7 ± 0.7
13.6 ± 0.5 17.6 ± 0.2
0.0 ± 0.0 0.0 ± 0.0
Errors are reported in term of standard deviation, * = P ≤ 0.05.
fatty acids in the biomass. Further, 1H spectrum of deuterated annual ryegrass exhibited the similar group of signals in the region from 3.0−5.5 ppm, while the signals corresponding to lignin aromatic protons were also observed in the 1H spectrum of deuterated annual ryegrass. 1H and 2H NMR spectra of control protiated ryegrass were also obtained to compare and ensure the deuteration (Figure 5C,D). The 1H spectrum of control protiated ryegrass exhibited all of the characteristic signals of biomass in the range of 3.0−5.5 ppm as well as also exhibited the aromatic proton signals corresponding to lignin component while the 2 H NMR did not show any corresponding detectable deuterium signals in that range, which confirmed the substantial deuterium incorporation in the rye grown in 50% D2O. Further, for the quantitative analysis, the concentration of proton nuclei in deuterated ryegrass in deuterated solvent was determined from the 1H NMR spectrum, with respect to an internal standard (TFA/d-TFA), while 2H NMR spectrum of the deuterated ryegrass dissolved in a protiated solvent indicated deuteron nuclei concentration with respect to the same internal standard (TFA/d-TFA). After
normalizing the concentration of deuterated rye in both solutions, the ratio of deuterons to protons (2H/1H) was calculated. The 2H/1H ratio indicated a deuterium incorporation of 36.9% in annual rye germinated and grown in 50% D2O. Growth rates of protiated ryegrass plants were doubled following transfer to the perfusion chambers while the growth rates of the plants transferred to 50% D2O did not change significantly from their growth in H2O before perfusion. A significant increase in the deuterium incorporation to ∼60% was achieved in the ryegrass that was germinated in H2O, then transferred to 50% D2O and grown inside the perfusion chambers. The seedlings started in H2O before transfer to 50% D2O appear to better tolerate high D2O concentrations than the plants grown from germination in 50% D2O, which could explain the substantially higher 2H incorporation. The current procedure thus offers a method to cultivate annual rye and similar plants to produced deuterated biomass with controlled deuterium incorporation for use in neutron scattering experiments. 2600
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Figure 6. FTIR spectra of deuterated ryegrass, protiated ryegrass and their delignified samples.
Table 5. Bands Assignments43,44 of FT-IR Spectra of Annual Ryegrass Samples
Carbohydrate and Klason Lignin Analysis. Table 4 shows the compositional analysis of deuterated and control protiated annual ryegrass samples and is comparable with the literature values.42 The data presented here are on an oven-dry (105 °C) basis and extractives were removed prior to each measurement. As Table 4 indicates, glucan and xylan were the predominant carbohydrates found in Lolium rye. The glucan content was slightly higher in deuterated annual ryegrass compared to that for control protiated annual ryegrass. The major hemicellulose found in ryegrass was xylan; however, small amounts of arabinan and galactan were also present. The total hemicellulose content of the deuterated ryegrass was slightly lower in comparison to that for control ryegrass. The relative klason lignin content was also lesser in the deuterated sample. However, there was not significant variation realized in the overall carbohydrates composition for both samples. FTIR Analysis. The FTIR spectra of deuterated annual ryegrass, protiated annual ryegrass and their delignified samples are presented in Figure 6. The C−D stretching in deuterated samples at 2139−2251 cm−1 indicated nonexchangeable deuterium incorporation in the carbohydrates components of annual ryegrass. The O−D stretching at 2486 cm−1 attributed to the proton-deuteron exchange on the biomass surface. The broad and strong peak at ∼3334 cm−1 is attributed to the hydroxyl group indicating strong hydrogen bonding. All of the samples exhibited vibrations at 2917−2854 cm−1, attributed to the C−H stretching and at 1378 and 1249 cm−1 corresponded to the C−H bending modes. IR bands centered at 1453 and 1118 cm−1 can be assigned as C−H symmetrical bending and C−O stretching of cellulose respectively. A nominal isotopic effect was observed in all of the C−D bands in the FTIR of deuterated samples (Table 5). For example, bands at 1375 cm−1, which is characteristic of symmetric C−H deformation of cellulose43,44 showed a slight shift toward 1326 cm−1 in deuterated annual rye sample. Similarly, IR band at 1460 cm−1 corresponding to CC stretching vibration of the aromatic ring44 also showed an isotopic shift in deuterated annual ryegrass (Table 5). Vibrations at 1053 and 1249 cm−1 are mainly attributed to the structural features related to
IR vibrations (ν) cm−1
deuterated rye
protiated rye
deuterated rye (delignified)
protiated rye (delignified)
νO−H (stretch) νCH2 (stretch) νC−H (OMe) νO−D (stretch) νC−D (stretch) νCO (acid/ ether) νCO (ketonic) νCC (stretch) νCH2 (sym.def.) νCH2 (bend)/ νO−D (def) νC−O νC−O−C (strectching) νC−H (out-of plane def)
3331(b,s)
3328(b,s)
3334(b,s)
3328(b,s)
2921(s) 2854(w) 2486(w)
2946(s) 2986(w)
2912(s)
2917(s)
2486(w)
2168(m)
2168(m)
1727(m)
1735(s)
1727(m)
1734(m)
1648(s)
1647(s)
1647(s)
1648(m)
1548(w)
1548(m)
1543(m)
1544(w)
1418(m)
1460(s)
1418(m)
1457(w)
1326(w)
1375(s)
1340(m)
1370(m)
1261(w) 1036(s)
1249(s) 1053(s)
1267(w) 1018(s)
1243(m) 1044(s)
802(w)
827(m)
701(w)
720(w)
carbohydrates and indicated isotopic shifts in deuterated samples. Molecular and Morphological Properties. In an effort to determine the effect of growing annual rye in D2O on the biomass key characteristics related to enzymatic hydrolysis,45,46 the important molecular properties of deuterated and control protiated ryegrass were compared. The molecular weight of cellulose and hemicellulose and cellulose crystallinity of annual ryegrass grown in 50% D2O and its protiated control are presented in Table 6. Deuterated rye cellulose had a higher weight average and number average molecular weight compared to its protiated control. Also, it displayed higher polydispersity index than protiated rye cellulose. The difference observed here is clearly attributed to the major deuterium 2601
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Table 6. Molecular Weight and Crystallinity of Annual Ryegrass Cellulose cellulose
a
hemicellulose
sample
Mn × 105 (g/mol)
Mw × 106 (g/mol)
PDIb
crystallinity index (%)
Mn × 104 (g/mol)
Mw × 104 (g/mol)
PDIb
protiated ryegrass deuterated ryegrass
1.4 ± 0.03 1.7 ± 0.04(ns)
1.8 ± 0.03 2.7 ± 0.03a
1.3 1.5
46.0 ± 2.0 43.0 ± 2.0
2.2 ± 0.01 2.3 ± 0.02
7.3 ± 0.01 7.6 ± 0.01
3.3 3.3
P ≤ 0.01, ns = not significant. bPDI = polydispersity index.
Figure 7. 13C−CPMAS spectra of cellulose I isolated from annual ryegrass sample crystallinity index = δ86−92/ δ80−92 × 100.
incorporation in the cellulose component of the biomass as also shown by the 2H NMR spectrum. In contrast, there was no change in the weight and number average molecular weight of hemicellulose isolated from 50% D2O annual ryegrass. Possibly the ryegrass grown in D2O may preferentially incorporate deuterium into cellulose compared to other polysaccharides. The crystallinity of cellulose isolated from deuterated ryegrass, as determined by 13CP/MAS (Figure 7) was ∼43% which is lower than the cellulose crystallinity of protiated ryegrass. The 13 C NMR spectrum of cellulose I isolated from deuterated ryegrass was similar, to that of control; however, at optimal signal-to-noise lower resolution was achieved for cellulose isolated from deuterated ryegrass (not shown). This is probably due to fewer protons available in deuterated ryegrass cellulose in comparison to protiated controls and consequently, the 13C CP/MAS NMR, which is the technique based on the magnetization transfer from protons to carbons, is expected to be less efficient for deuterated substrates than for protonated substrates due to the differences in bond energy between protons and deuterons. Therefore, the subsequent enhancement of the 13C signal will be lessened in 50% D2O grown rye. Nonetheless, deuteration did not appear to cause any noticeable or significant difference in the cellulose crystallinity. Morphology of Deuterated Annual Ryegrass. The present paper attempts to study the surface morphology of D2O grown rye and demonstrates that no major disruption of micro fibrils was observed in the native surface of the rye grown in 50% D2O compared to its protiated counterpart. The SEM images of control protiated and deuterated annual ryegrass before and after the peracetic acid delignification process are shown in Figure 8A−D. Both the protiated and deuterated rye samples exhibited an elongated and well-ordered surface (Figure 8A,B). The texture was compact and shielded in a thin wax layer commonly found in herbaceous biomass.47 However, several subtle differences were observed in the deuterated rye leaf such as the deuterated rye leaf exhibited a surface layer with more debris and some noticeable granular structures on the individual fibers. Furthermore, the delignified surface of both samples was examined in an effort to find the effect of deuteration more closely on the morphological
Figure 8. SEM images of (A) deuterated ryegrass, (B) control protiated ryegrass, (C) deuterated ryegrass after delignification, and (D) protiated ryegrass after delignification.
structures in both rye samples. Surface morphology of both samples changed significantly during the delignification process. In both delignified samples the overall structure was disrupted (Figure 8C,D) and the individual fibers were partially separated and dispersed. Peracetic pulping removed most of the surface layer deposits, which were noticed in the native samples and consequently exposed intact cellulose microfibrils. In a delignified deuterated ryegrass, most of the leaf surface was degraded although it showed the cellulose micro fibrils partly embedded in the noncellulosic polymers as revealed by the rough and uneven surface (Figure 8C). The study demonstrates that the leaf morphology appeared to be basically similar for the two annual ryegrass samples, but with certain differences apparent, particularly following delignification. The biomass characterization results indicated some significant changes in the biomass structure with deuteration. There was an increase in the molecular weights of cellulose, whereas hemicellulose molecular weights and cellulose crystallinity remain unaffected with the deuteration. Nevertheless, the results support the development and use of deuterated plant biomass to provide 2602
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crucial supporting characterization for visualization of lignocellulose structure by neutron scattering techniques and computational simulation.
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AUTHOR INFORMATION
Corresponding Author
*Tel. 404-894-9701. Fax 404-894-4778. E-mail: arthur.
[email protected];
[email protected]. edu. Present Address ∥
University of Massachusetts, Amherst, Massachusetts.
Funding
This research was supported by the Genomic Science Program, Office of Biological and Environmental Research, U.S. Department of Energy, under Contract FWP ERKP752. The research at Oak Ridge National Laboratory’s Center for Structural Molecular Biology (CSMB) was supported by the Office of Biological and Environmental Research under Contract FWP ERKP291, using facilities supported by the Office of Basic Energy Sciences, U.S. Department of Energy. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract DEAC05-00OR22725. D.R. was supported by a U.S. Department of Energy Higher Education Research Experience internship managed by Oak Ridge Institute of Science and Education. Notes
The authors declare the following competing financial interest(s): The author receives research funding in this field of study.
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