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Main Conclusion: Common duckweed Lemna minor was cultivated in 50% D 2 O to produce biomass with 50-60% deuterium incorporation containing cellulose with degree of polymerization close (85%) to that of H 2 O-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% D 2 O with 0.5% glucose. L. minor grown in 50% D 2 O without addition of kinetin exhibited a lag phase twice that of H 2 O-grown controls, before start of log phase growth at 40% of control rates. Compared to continuous white fluorescent light, growth rates increased fivefold for H 2 O and twofold for 50% D 2 O 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 1 H and 2 H nuclear magnetic resonance (NMR) to be 40-60%. The cellulose from the deuterated plants had an average-number degree of polymerization (DP n ) and polydispersity index (PDI) close to that of H 2 O-grown controls, while Klason lignin content was reduced. The only major gross morphological change noted was root inhibition.

Main Conclusion: 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.

In vivo generated deuterated bacterial cellulose, cultivated from 100% deuterated glycerol in D2O medium, was analyzed for deuterium incorporation by ionic liquid dissolution and (2)H and (1)H nuclear magnetic resonance (NMR). A solution NMR method of the dissolved cellulose was used to determine that this bacterial cellulose had 85% deuterium incorporation. Acetylation and (1)H and (2)H NMR of deuterated bacterial cellulose indicated near equal deuteration at all sites of the glucopyranosyl ring except C-6 which was partly deuterated. Despite the high level of deuterium incorporation no significant differences in the molecular and morphological properties were observed for the deuterated and protio bacterial cellulose samples. The highly deuterated bacterial cellulose presented here can be used as a model substrate for studying cellulose biopolymer properties via future small angle neutron scattering (SANS) studies., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A commercially available deuterated kale sample was analyzed for deuterium incorporation by ionic liquid solution (2)H and (1)H nuclear magnetic resonance (NMR). This protocol was found to effectively measure the percent deuterium incorporation at 33%, comparable to the 31% value determined by combustion. The solution NMR technique also suggested by a qualitative analysis that deuterium is preferentially incorporated into the carbohydrate components of the kale sample.