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Common duckweed Lemna minor was cultivated in 50% D2O to produce biomass with 50–60% deuterium incorporation containing cellulose with degree of polymerization close (85%) to that of H2O-grown controls. The small aquatic plant duckweed, particularly the genus Lemna, widely used for toxicity testing, has been proposed as a potential source of biomass for conversion into biofuels as well as a platform for production of pharmaceuticals and specialty chemicals. Ability to produce deuterium-substituted duckweed can potentially extend the range of useful products as well as assist process improvement. Cultivation of these plants under deuterating conditions was previously been reported to require addition of kinetin to induce growth and was hampered by anomalies in cellular morphology and protein metabolism. Here, we report the production of biomass with 50–60% deuterium incorporation by long-term photoheterotrophic growth of common duckweed Lemna minor in 50% D2O with 0.5% glucose. L. minor grown in 50% D2O without addition of kinetin exhibited a lag phase twice that of H2O-grown controls, before start of log phase growth at 40% of control rates. Compared to continuous white fluorescent light, growth rates increased fivefold for H2O and twofold for 50% D2O when plants were illuminated at higher intensity with a metal halide lamp and a diurnal cycle of 12-h light/12-h dark. Deuterium incorporation was determined by a combination of 1H and 2H nuclear magnetic resonance (NMR) to be 40–60%. The cellulose from the deuterated plants had an average-number degree of polymerization (DPn) and polydispersity index (PDI) close to that of H2O-grown controls, while Klason lignin content was reduced. The only major gross morphological change noted was root inhibition.

Common duckweed Lemna minor was cultivated in 50% D

Main conclusion The bioenergy crop switchgrass was grown hydroponically from tiller cuttings in 50 % D2O to obtain biomass with 34 % deuterium substitution and physicochemical properties similar to those of H2O-grown switchgrass controls. Deuterium enrichment of biological materials can potentially enable expanded experimental use of small angle neutron scattering (SANS) to investigate molecular structural transitions of complex systems such as plant cell walls. Two key advances have been made that facilitate cultivation of switchgrass, an important forage and biofuel crop, for controlled isotopic enrichment: (1) perfusion system with individual chambers and (2) hydroponic growth from tiller cuttings. Plants were grown and maintained for several months with periodic harvest. Photosynthetic activity was monitored by measurement of CO2 in outflow from the growth chambers. Plant morphology and composition appeared normal compared to matched controls grown with H2O. Using this improved method, gram quantities of switchgrass leaves and stems were produced by continuous hydroponic cultivation using growth medium consisting of basal mineral salts in 50 % D2O. Deuterium incorporation was confirmed by detection of the O-D and C-D stretching peaks with FTIR and quantified by 1H- and 2H-NMR. This capability to produce deuterated lignocellulosic biomass under controlled conditions will enhance investigation of cell wall structure and its deconstruction by neutron scattering and NMR techniques.

Exogenous phenylalanine stunted annual ryegrass but not switchgrass or winter grain rye, with deuterium incorporation up to 3% from phenyalanine-d 8 . Toxicity to duckweed varied with illumination intensity and glucose uptake. Isotopic labeling of biomolecules through biosynthesis from deuterated precursors has successfully been employed for both structural studies and metabolic analysis. Phenylalanine is the precursor of many products synthesized by plants, including the monolignols used for synthesis of lignin. Possible allelochemical effects of phenylalanine have not been reported, although its deamination product cinnamic acid is known to have deleterious effects on root elongation and growth of several plant species. The effects of phenylalanine and its deuterated analog phenylalanine-d 8 added to growth media were studied for annual ryegrass (Lolium multiflorum), winter grain rye (Secale cereale), and switchgrass (Panicum virgatum) cultivated under hydroponic conditions. Growth of annual ryegrass was inhibited by phenylalanine while switchgrass and rye were not significantly affected. Growth was less affected by deuterated phenylalanine-d 8 than by its protiated counterpart, which may be a typical deuterium kinetic isotope effect resulting in slower enzymatic reaction rates. Deuterium incorporation levels of 2–3% were achieved in biomass of switchgrass and annual ryegrass. Both protiated and deuterated phenylalanine were moderately toxic (IC25 values 0.6 and 0.8 mM, respectively) to duckweed (Lemna minor) grown using a 12 h diurnal cycle under photoautotrophic conditions. A significant increase in toxicity, greater for the deuterated form, was noted when duckweed was grown under higher intensity, full spectrum illumination with a metal halide lamp compared to fluorescent plant growth lamps emitting in the blue and red spectral regions. Supplementation with glucose increased toxicity of phenylalanine consistent with synergy between hexose and amino acid uptake that has been reported for duckweed.