Supplementary Information for:
Production of deuteratedswitchgrassby hydroponic cultivation
For submission to the journal
Planta
Barbara R. Evans,a Garima Bali,b Marcus Foston,b§ Arthur J.Ragauskas,b* Hugh O’Neill,c Riddhi Shah,c Joseph McGaughey,a҂ David Reeves,a‡ Caroline Rempe,a# and Brian H. Davisond
aChemical Sciences Division, Oak Ridge National Laboratorye, Oak Ridge, TN, 37831 U.S.A. Corresponding author e-mail Tel. 1-865-241-3185, Fax 1-865-574-4961
bInstitute of Paper Science and Technology, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332 U.S.A.
cBiology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 U.S.A.
dBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 U.S.A.
eThis manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.
§ Current address Department of Energy, Environmental & Chemical Engineering, Washington University, St. Louis, MO 63130, USA.
*Current address Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, 37996, and Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831
҂ Current address 5105 Greentree Drive, Nashville, TN 37211, USA.
‡ Current address Department of Languages, Literatures, and Cultures, University of Massachusetts, Amherst, MA, 01003, USA.
# Current address School of Genome Science and Technology, F337 Walters Life Science, University of Tennessee, Knoxville, 37996, TN, USA.
This SI document in the following 9 pages (S2– S10) includes text, tables, and figures with detail on:
(1) Plant Cultivation
(2) Analytical Methods
(3) Structural Characterization
(1)Plant Cultivation
Materials
Switchgrass seeds (Panicumvirgatum, Alamo cultivar) were obtained from the Bioenergy Science Center located at Oak Ridge National Laboratory, Oak Ridge, Tennessee. Deuterium oxide (D2O), 99.8%, was purchased from Cambridge Isotope Laboratories (Cambridge, Massachusetts). All plant growth solutions except for those used for the tissue culture experiments were made with Schenk and Hildebrandt’s basal salts (Phytotechnology Laboratories, Shawnee Mission, Kansas). Composition of Schenk and Hildebrandt’s basal salts prepared at3.2 g L-1 is given by the manufacturer as: 300 mg/L ammonium phosphate monobasic, 5 mg/L boric acid, 151 mg/L anhydrous calcium chloride, 0.1 mg/L cobalt chloride hexahydrate, 0.2 cupric sulfate pentahydrate, 20 mg/L Na2EDTA dihydrate, 15 ferrous sulfate heptahydrate, 195.4 mg/L anhydrous magnesium sulfate, 10 mg/L manganese 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. No vitamins or hormones were added to the plant growth solutions used for cultivation in soil or hydroponic solutions. Plant jars, unvented and 0.2 µ filter vented polypropylene lids, agar, Murashige and Skoop media, and plant hormones used for tissue culture experiments were purchased from Phytotechnology Laboratories (Shawnee Mission, Kansas, U. S. A.). Distilled water was further purified with a MilliQ (Millipore), NanoDiamond (Barnstead), or E-Pure (Barnstead Thermolyne, Dubuque, Iowa, U. S. A.) water purification system before use. All solutions, equipment, and soil were steam-sterilized in an autoclave before use. Solutions containing D2O were filter-sterilized.
Illumination was provided by a metal halide lamp mounted in a SunSystem 2 lamp holder (Future Garden, North Lindhurst, New York, U. S. A.) that was suspended from an in-house built lamp stand and Sylvania Gro-Lamps mounted on a plant cart (Carolina Biological Supply Company, Burlington, North Carolina, U. S. A.). Illumination intensity was 150 µmol photons m-2 s-1measured with an LI-250 Light Meter (LI-COR, Lincoln, Nebraska, U. S. A.) and 8930luxmeasured with a Traceable Light Meter (VWR International LLC, Radnor, Pennsylvania, U. S. A.). Long-wavelength UV light (300 – 400 nm) was measured with a UVP J221 UV light meter. Short-wavelength UV (295-325 nm) was measured with aErythema No. 1638 meter (Carlton Industries). Diurnal cycle was 12 hours light, 12 hours dark.
Seed Germination
Switchgrass seeds were germinated in commercial seed starting products that were steam-sterilized before use in perfusion chamber experiments to avoid fungal and algal contamination. Potting soil (Fafard seed starter mix, Conrad Fafard, Inc., Agawam, Massachusetts, U. S. A.)was used in open pots or sterilized in glass jars. Jiffy Peat Pellets (Jiffy Products of America Inc., Lorain, Ohio, U.S.A.)were inserted into 2-inch plastic hydroponic baskets in glass plant jars (16 and 32 oz.). The pellets were swollen in solutions of Schenk and Hildebrandt’s basal salts before steam sterilization. Seeds were surface-sterilized with 70% ethanol, followed by soaking in Schenk & Hildebrandt’s basal salts in H2O before planting.Stratified seeds were treated the same except they were stored moist for 14 days at 8 °C before planting.Stratification of seed by cold treatment occasionally improved and speeded germination, but results between trials varied, consistent with published reports(Bouton 2008; Douglas et al. 2009; Zegada-Lizarazu et al. 2012). Surface-sterilization with 0.4% sodium hypochlorite (5% dilution of double strength Clorox bleach) in sterile distilled water for 20 min, followed by three water washes and imbibition in Schenk and Hildebrandt’s basal salts for 4 – 72 h proved to be more effective than cold stratification or direct planting. Switchgrass plants in peat pellets were transferred to perfusion chambers once seedlings reached 15 cm height. Seedlings were grown in soil watered with H2O for two to three months until secondary shoots (tillers) were established before use in experiments.
Tissue culture
Plant tissue culture was carried out according to published methods developed for switchgrass propagation from stem sections and callus tissue (Zegada-Lizarazu et al. 2012; Alexandrova et al. 1996; Conger 2002). Complete Murashige and Skoop medium thickened with 0.5% plant agar and supplemented with 2,4-D (2, 4-dichloroindoleacetic acid) and 3-BAP (3-benzylaminopurine) was used to prepare agar plates and stabs. Stem sections were taken from young shoots that had been surface-sterilized with 70% ethanol.
Hydroponic perfusion chambers
The multiple-chamber system for simultaneous cultivation of several plants in isolated chambers under perfusion with dry air has been described previously(Evanset al. 2014). Briefly, perfusion chambers were assembled from 1-liter graduated glass cylinders closed with two-hole rubber stoppers. Air flow was provided by glass tubing inserted into the stoppers. The outflow tubes were connected to glass condensation coils to limit evaporation loss. Sterile 0.22 micron 33 mm diameter syringe filters (Millipore Ireland Ltd., Tullagreen, Carrigtwohill, Cork, Ireland) were attached to both outflow and inflow tubing to prevent microbial contamination. The outflow from each deuteration chambers was routed through a glass water trap cooled to 6 °C by a refrigerated circulating water bath in order to condense 50% D2O-water vapor for recycle. Captured and recycled 50% D2O comprised 20% of the total volume of growth solution used to maintain the switchgrass plants. The chambers were perfused with ambient air dried with Eagle silica gel desiccant using Marina 50 aquarium pumps [Rolf Hagen (USA Corp.), Mansfield, Massachusetts, U.S.A.]. Carbon dioxide concentration of the dried, filtered ambient air was determined to be 0.04% by measurement with an LI-6252 CO2 Analyzer (LI-COR, Lincoln, Nebraska, U. S. A.) that was calibrated with analyzed air mixtures air zero and 698 ppm CO2 in air (Air Liquide, Oak Ridge, Tennessee, U.S.A.). Air flow rates were measured with an Alltech Digital Flowcheck HR meter. Growth medium was Schenk and Hildebrandt’s basal salts (Phytotechnology Laboratories, Shawnee Mission, Kansas, U.S.A.).
For vegetative propagation, shoots were cut below the junction with the rhizome crown and trimmed to 8 cm long, then inserted into supports in baskets and grown in Schenk & Hildebrandt’s basal salts in H2O in plant jars. Glass fibre filters (Millipore Ireland Ltd., Tullagreen, Carrigtwohill, Cork, Ireland) were used as supports for the cuttings inside the baskets. Successfully rooted cuttings were transferred to individual 2-inch hydroponic baskets and put into perfusion chambers. Plant establishment by hydroponic growth of tiller cuttings was compared to seed germination in potting soil and peat plugs (Table S-1). Successful rooting and growth of tiller cuttings was significantly greater than that of seed germination in either potting soil or in peat plugs (Tables S-1 and S-2). Growth rates of the tiller cuttings during the first month of growth were also significantly higher than those of young seedlings germinated in potting soil or peat plugs (Tables S-1 and S-3). There was no significant difference between the two soil types used, potting soil and peat plugs, for germination and initial growth rates of switchgrass (Tables S-1, S-2, and S-3).
Leaves and stalks were periodically harvested from tillers for analysis and to allow continued growth (Tables S-4 and S-5), every one to four months. As the tops of the stalks and leaves reached the lids of the perfusion chambers, the tillers continued to grow, folding over to adapt to the chamber height restrictions (about 35 cm). At harvest, the lids were removed and the leaves stretched out and measured (Table S-5). For the 1-liter perfusion chambers, the average heights of the switchgrass tillers grown in 50% D2O were not statistically different from those of hydroponic switchgrass grown in H2O (P > 0.5), and the maximum observed height in 50% D2O was 91% of that of the highest H2O tiller. Heights of hydroponic H2O-grown switchgrass tillers were not statistically different than those of switchgrass grown in peat plugs from seed in H2O in the 1-liter perfusion chambers (P > 0.1).
The perfusion chamber design enables non-destructive monitoring of photosynthetic activity by measurement of gases in the air outflow from individual chambers. Photosynthetic activity was estimated by monitoring CO2 concentrations in the air outflow from the perfusion chambers and calculating the difference from the concentration in the air feed (Table S-6). Uptake rates were normalized to the total tiller heights of each plant to enable comparison between plants with differing number and sizes of tillers.Uptake of CO2 by a hydroponic switchgrass plant growing in 50% D2O was 82% of that of a hydroponic switchgrass plant growing in H2O.
Table S-1. Plant establishment and growth rates during the first month of growth are compared for germination from seed in soil and for vegetative propagation from cuttings in hydroponic solution.
Growth matrix / Propagation / Seedlings/trial (%) / Growth rate (cm/d)Potting soil / Seed / 6.8 ± 3.5 (N = 6) / 0.57 ± 0.16 (N=6)
Peat pellet / Seed / 5.6 ± 4.5 (N =4) / 0.73 ± 0.06 (N = 3)
Hydroponic / Cutting / 45.8 ± 16.7 (N=6) / 1.1 ± 0.31(N = 8)
Table S-2. P values were calculated with the Student t-test for the plant establishment data in Table S-1 to determine the statistical significance.
Propagation (growth matrix) / Seed (soil) / Seed (peat pellet) / Vegetative (hydroponic)Seed (soil) / 1 / 0.70 / 0.003
Seed (peat pellet) / 0.70 / 1 / 0.002
Vegetative (hydroponic) / 0.003 / 0.002 / 1
Table S-3. P values were calculated with the Student t-test to determine the statistical significance of the comparative growth rates during first month given in Table S-1.
Growth Matrix / Potting soil / Peat pellet / HydroponicPotting soil / 1 / 0.10 / 0.004
Peat pellet / 0.10 / 1 / 0.02
Hydroponic / 0.004 / 0.02 / 1
TableS-4.Stem and leaf samples were periodically harvested from a switchgrass plant established from a tiller cutting in hydroponic culture during initial growth in H2O, and after transfer to 50% D2O solution.
Cultivation Step / Cumulative Growth Time (Days) / Net Growth Time(Days) / Fresh Weight (g)
Establishment in H2O / 67 / 67 / 0.4577
127 / 60 / 0.1551
Growth in 50% D2O / 36 / 36 / 0.5174
84 / 48 / 1.6138
172 / 88 / 1.9532
227 / 55 / 1.3055
267 / 40 / 0.8135
322 / 55 / 3.5125
344 / 22 / 2.2541
373 / 29 / 2.0835
431 / 58 / 4.0553
466 / 35 / 3.6576
487 / 21 / 2.7350
500 / 13 / 3.6177
Table S-5.Comparison of tiller heights and number at bimonthly harvest for hydroponic switchgrass grown in perfusion chambers in H2O and in 50% D2O, and for switchgrassstarted from seed and grown in peat plugs in H2O in the perfusion chambers.
Growth condition / Average height (cm) / Maximum height (cm) / Number of tillersH2O hydroponic / 49.6 ± 22.5 / 82 / 8
50% D2O hydroponic / 48.95 ± 10.3 / 75 / 10
H2O peat plug / 60.3 ± 16.8 / 74 / 8
Table S-6. Comparison of photosynthetic activity of switchgrass plants growing in perfusion chambers as measured by the difference between carbon dioxideconcentrations in the air outflow from each chamber and the concentration (403 ppm) in the air feed.
Cultivation description / Tiller height (cm) / Total height (cm) / CO2 out (ppm) / Air flow (ml/min) / Net CO2 uptake (ppm) / Net CO2 uptake (µmolmin-1) / Net CO2 uptake normalized to tiller height (µmolmin-1cm-1)Germinationpeat plug H2O / 27; 55 / 82 / 33 / 40 / 370 / 0.609 / 0.00743
Vegetative hydroponic H2O / 15.5; 19 / 34.5 / 154 / 41 / 249 / 0.420 / 0.0122
Vegetative hydroponic 50% D2O / 36 / 36 / 38 / 24 / 365 / 0.360 / 0.0103
(2)Analytical Methods
Deuterium incorporation was estimated by FTIR analysis of leaf and stem samples and quantified by NMR as described previously (Foston et al. 2012; Bali et al. 2013; Evans et al. 2014). Infrared (FTIR) spectra were obtained with 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 cm-1 to 500 cm-1. After the base-line correction, the spectra were scaled with respect to one another, using the C–O stretching of the glucopyranose ring at ~1032 cm−1.
Table S-7.FTIR band assignments (Ding et al. 2112; Shih and Li 2012) used for identification in Figure 4.
b:broad, s: strong, w:weak m:medium
3) Structural Characterization
Compositional analysis
Samples for carbohydrate and acid-insoluble lignin analysis were prepared using a two-stage acid hydrolysis protocol based on Tappi method T-222 om-88. 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., Sunnyvale, California, USA). TableS8shows the compositional analysis of hydroponicswitchgrass samples. The data presented here are calculated on an oven-dry (105°C) basis and extractives were removed prior to each measurement. As shown inTable S8, glucan and xylan were the major carbohydrates present in both switchgrasssamples with minor amounts of arabinan, galactanand mannan. The total hemicellulose content of the switchgrass grown in D2O was not statistically different thanthe control sample by Student’s T-test(P =0.1to 0.4 for each hemicellulose components except arabinan for which P is 0.02; If P≥0.05 then the difference is not considered significant). Glucancontent in hydroponic 50% D2O-grown switchgrasswaslower than that of switchgrass grown in H2O, but not statistically different(P = 0.09) by Student’s T-test.
Table S-8. Compositional Analysisa of Hydroponic Switchgrass Compared to Switchgrass Grown from Seed in a Peat Plug.
aPercentages based on dry biomass. The standard deviations associated with lignin and carbohydrate analysis were 0.5 - 0.7 and 0.01-1.54, respectively.
bExtractives, ash, uronic acids etc.
Samples / Glucan (%) / Xylan (%) / Arabinan (%) / Galactan (%) / Mannan (%) / Lignin % / Otherb (%)Peat plug H2O / 39.46 / 18.28 / 3.78 / 1.46 / 0.17 / 11.24 / 26
Hydroponic H2O / 36.58 / 16.06 / 3.45 / 1.39 / 0.15 / 9.29 / 33
Hydroponic 50% D2O / 34.79 / 16.10 / 3.65 / 1.37 / 0.14 / 16.00 / 28
Literature References
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Bouton JH, Chapter 11, Improvement of Switchgrass as a Bioenergy Crop, In Genetic Improvement of Bioenergy Crops, W. Vermerris, ed., Springer Science + Business Media, LLC, pp. 295 – 308 (2008).
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