Comparison among Activities and Isoflavonoids from Pueraria thunbergiana Aerial Parts and Root.

: Kudzu (Pueraria thunbergiana Benth.) has long been used as a food and medicine for many centuries. The root is the most commonly used portion of the plant, but the aerial parts are occasionally used as well. In this study, we investigated the constituent compounds and biological activities of the aerial parts, leaves, stems, and sprouts, and compared their constituents and activities with those of roots. Leaf extract showed a significantly higher TPC level at 59 ± 1.6 mg/g and lower free radical scavenging (FRS) values under 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and NO inhibition at 437 ± 11, 121 ± 6.6 µg/mL and 107 ± 4.9 µg/mL, respectively, than those of sprout, stem, and root extract. Leaf extract also significantly suppressed lipopolysaccharide (LPS)-mediated gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The main components of leaf extract were found to be genistin and daidzin. This study suggests that the leaves of kudzu are a good source of biological activities and isoflavones that can be used in functional or medicinal foods and cosmetics for the prevention or treatment of diseases related to inflammation and oxidative stress.

Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables.

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.

Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF.

BACKGROUND: Construction of microbial biocatalysts for the production of biorenewables at economically viable yields and titers is frequently hampered by product toxicity. Membrane damage is often deemed as the principal mechanism of this toxicity, particularly in regards to decreased membrane integrity. Previous studies have attempted to engineer the membrane with the goal of increasing membrane integrity. However, most of these works focused on engineering of phospholipids and efforts to identify membrane proteins that can be targeted to improve fatty acid production have been unsuccessful. RESULTS: Here we show that deletion of outer membrane protein ompF significantly increased membrane integrity, fatty acid tolerance and fatty acid production, possibly due to prevention of re-entry of short chain fatty acids. In contrast, deletion of fadL resulted in significantly decreased membrane integrity and fatty acid production. Consistently, increased expression of fadL remarkably increased membrane integrity and fatty acid tolerance while also increasing the final fatty acid titer. This 34% increase in the final fatty acid titer was possibly due to increased membrane lipid biosynthesis. Tuning of fadL expression showed that there is a positive relationship between fadL abundance and fatty acid production. Combinatorial deletion of ompF and increased expression of fadL were found to have an additive role in increasing membrane integrity, and was associated with a 53% increase the fatty acid titer, to 2.3 g/L. CONCLUSIONS: These results emphasize the importance of membrane proteins for maintaining membrane integrity and production of biorenewables, such as fatty acids, which expands the targets for membrane engineering.

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity.

Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.

Metabolic flux analysis of Escherichia coli MG1655 under octanoic acid (C8) stress.

Systems metabolic engineering has made the renewable production of industrial chemicals a feasible alternative to modern operations. One major example of a renewable process is the production of carboxylic acids, such as octanoic acid (C8), from Escherichia coli, engineered to express thioesterase enzymes. C8, however, is toxic to E. coli above a certain concentration, which limits the final titer. (13)C metabolic flux analysis of E. coli was performed for both C8 stress and control conditions using NMR2Flux with isotopomer balancing. A mixture of labeled and unlabeled glucose was used as the sole carbon source for bacterial growth for (13)C flux analysis. By comparing the metabolic flux maps of the control condition and C8 stress condition, pathways that were altered under the stress condition were identified. C8 stress was found to reduce carbon flux in several pathways: the tricarboxylic acid (TCA) cycle, the CO2 production, and the pyruvate dehydrogenase pathway. Meanwhile, a few pathways became more active: the pyruvate oxidative pathway, and the extracellular acetate production. These results were statistically significant for three biological replicates between the control condition and C8 stress. As a working hypothesis, the following causes are proposed to be the main causes for growth inhibition and flux alteration for a cell under stress: membrane disruption, low activity of electron transport chain, and the activation of the pyruvate dehydrogenase regulator (PdhR).

Monodisperse pattern nanoalloying for synergistic intermetallic catalysis.

Nanoscale alloys attract enormous research attentions in catalysis, magnetics, plasmonics and so on. Along with multicomponent synergy, quantum confinement and extreme large surface area of nanoalloys offer novel material properties, precisely and broadly tunable with chemical composition and nanoscale dimension. Despite substantial progress of nanoalloy synthesis, the randomized positional arrangement and dimensional/compositional inhomogeneity of nanoalloys remain significant technological challenges for advanced applications. Here we present a generalized route to synthesize single-crystalline intermetallic nanoalloy arrays with dimensional and compositional uniformity via self-assembly. Specific electrostatic association of multiple ionic metal complexes within self-assembled nanodomains of block copolymers generated patterned monodisperse bimetallic/trimetallic nanoalloy arrays consisting of various elements, including Au, Co, Fe, Pd, and Pt. The precise controllability of size, composition, and intermetallic crystalline structure of nanoalloys facilitated tailored synergistic properties, such as accelerated catalytic growth of vertical carbon nanotubes from Fe-Co nanoalloy arrays.

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition.

Soybean [Glycine max (L.) Merr.] seed are valued for their protein and oil content. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. This report describes the growth dynamics and compositional changes of SHaM embryos in response to change in the carbon to nitrogen ratio of the medium. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates, embryo protein content was strongly correlated with decreasing C:N ratios and increasing glutamine consumption rates. However, oil content remained relatively unchanged across the C:N ratio range tested, and resources were instead directed towards the starch and residual biomass (estimated by mass balance) pools in response to increasing C:N ratios. Protein and oil were inversely related only at concentrations of sucrose in the medium <88 mM, where carbon limited growth and no starch was found to accumulate in the tissues. These observations and the high reproducibility in the data indicate that SHaM embryos are an ideal model system for the application of metabolic flux analysis studies designed to test hypotheses regarding assimilate partitioning in developing soybean seeds.

Self-assembly-induced formation of high-density silicon oxide memristor nanostructures on graphene and metal electrodes.

We report the direct formation of ordered memristor nanostructures on metal and graphene electrodes by a block copolymer self-assembly process. Optimized surface functionalization provides stacking structures of Si-containing block copolymer thin films to generate uniform memristor device structures. Both the silicon oxide film and nanodot memristors, which were formed by the plasma oxidation of the self-assembled block copolymer thin films, presented unipolar switching behaviors with appropriate set and reset voltages for resistive memory applications. This approach offers a very convenient pathway to fabricate ultrahigh-density resistive memory devices without relying on high-cost lithography and pattern-transfer processes.

An integrated computational and experimental study for overproducing fatty acids in Escherichia coli.

Increasing demands for petroleum have stimulated sustainable ways to produce chemicals and biofuels. Specifically, fatty acids of varying chain lengths (C6-C16) naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids from plants and other microbial production hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways. In addition, precursors for fatty acids are used along other central metabolic pathways for the production of amino acids and biomass, which further complicates the engineering of microbial hosts for higher yields. Here, we demonstrate an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis techniques to meet this challenge. The OptForce procedure was used for suggesting and prioritizing genetic manipulations that overproduce fatty acids of different chain lengths from C6 to C16 starting with wild-type E. coli. We identified some common but mostly chain-specific genetic interventions alluding to the possibility of fine-tuning overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose (∼39% maximum theoretical yield) of C14₋16 fatty acids in minimal M9 medium. These results highlight the benefit of using computational strain design and flux analysis tools in the design of recombinant strains of E. coli to produce free fatty acids.

Fabrication of high-density In(3)Sb(1)Te(2) phase change nanoarray on glass-fabric reinforced flexible substrate.

Mushroom-shaped phase change memory (PCM) consisting of a Cr/In(3)Sb(1)Te(2) (IST)/TiN (bottom electrode) nanoarray was fabricated via block copolymer lithography and single-step dry etching with a gas mixture of Ar/Cl(2). The process was performed on a high performance transparent glass-fabric reinforced composite film (GFR Hybrimer) suitable for use as a novel substrate for flexible devices. The use of GFR Hybrimer with low thermal expansion and flat surfaces enabled successful nanoscale patterning of functional phase change materials on flexible substrates. Block copolymer lithography employing asymmetrical block copolymer blends with hexagonal cylindrical self-assembled morphologies resulted in the creation of hexagonal nanoscale PCM cell arrays with an areal density of approximately 176 Gb/in(2).

Expression of glutathione S-transferases in poplar trees (Populus trichocarpa) exposed to 2,4,6-trinitrotoluene (TNT).

Twelve Populus genes were identified from Arabidopsis thaliana sequences previously shown to be induced by exposure to 2,4,6-trinitrotoluene (TNT). Using the resources of the Poplar Genome Project and National Center for Biotechnology Information databases, Populus conserved domains were identified and used to design gene specific primers. RNA extracted from root tissues of TNT-exposed hydroponic poplar plants was used to quantify the expression of genes by reverse-transcriptase real-time polymerase chain reaction. Cyclophilin and 18S ribosomal DNA genes were used as internal standards. Exposure to TNT resulted in a significant increase of gene expression of two glutathione S-transferases (GST), peaking at levels of 25.0 +/- 13.1 and 10 +/- 0.7 fold the expression level of non-exposed plants after 24 h for each of the GST genes, respectively. This paper demonstrates the use of functional genomics information from the model plant species, Arabidopsis, to identify genes which may be important in detoxification of TNT in the model phytoremediation species, Populus trichocarpa.

Engineering of E. coli inherent fatty acid biosynthesis capacity to increase octanoic acid production.

BACKGROUND: As a versatile platform chemical, construction of microbial catalysts for free octanoic acid production from biorenewable feedstocks is a promising alternative to existing petroleum-based methods. However, the bio-production strategy has been restricted by the low capacity of E. coli inherent fatty acid biosynthesis. In this study, a combination of integrated computational and experimental approach was performed to manipulate the E. coli existing metabolic network, with the objective of improving bio-octanoic acid production. RESULTS: First, a customized OptForce methodology was run to predict a set of four genetic interventions required for production of octanoic acid at 90% of the theoretical yield. Subsequently, all the ten candidate proteins associated with the predicted interventions were regulated individually, as well as in contrast to the combination of interventions as suggested by the OptForce strategy. Among these enzymes, increased production of 3-hydroxy-acyl-ACP dehydratase (FabZ) resulted in the highest increase (+ 45%) in octanoic acid titer. But importantly, the combinatorial application of FabZ with the other interventions as suggested by OptForce further improved octanoic acid production, resulting in a high octanoic acid-producing E. coli strain +fabZ ΔfadE ΔfumAC ΔackA (TE10) (+ 61%). Optimization of TE10 expression, medium pH, and C:N ratio resulted in the identified strain producing 500 mg/L of C8 and 805 mg/L of total FAs, an 82 and 155% increase relative to wild-type MG1655 (TE10) in shake flasks. The best engineered strain produced with high selectivity (> 70%) and extracellularly (> 90%) up to 1 g/L free octanoic acid in minimal medium fed-batch culture. CONCLUSIONS: This work demonstrates the effectiveness of integration of computational strain design and experimental characterization as a starting point in rewiring metabolism for octanoic acid production. This result in conjunction with the results of other studies using OptForce in strain design demonstrates that this strategy may be also applicable to engineering E. coli for other customized bioproducts.

Phytotoxicity and phytoremediation of 2,6-dinitrotoluene using a model plant, Arabidopsis thaliana.

Biochemical and genetic studies of xenobiotic metabolism in the model plant Arabidopsis have significant potential in providing information for phytoremediation. This paper presents the toxicity of 2,6-dinitrotoluene (2,6-DNT) to Arabidopsis under axenic conditions, the fate and transformation of 2,6-DNT after uptake by the plant, and the effect of a putative glutathione S-transferase (GST), which is highly induced by 2,4,6-trinitrotoluene (TNT) in the previous study, on the detoxification of 2,6-DNT. 2,6-DNT had toxic effects on the growth of Arabidopsis based on whole seedling as well as root growth assays. Using [U- 14C]2,6-DNT, the recovery was over 87% and less than 2% accounted for the mineralization of 2,6-DNT in axenic liquid cultures during the 14d of exposure. About half (48.3%) of the intracellular radioactivity was located in the root tissues in non-sterile hydroponic cultures. 2-Amino-6-nitrotoluene (2A6NT) and two unknown metabolites were produced as transformation products of 2,6-DNT in the liquid media. The metabolites were further characterized by proton NMR spectra and the UV-chromatograms when the plant was fed with either 2,6-DNT or 2A6NT. In addition, polar unknown metabolites were detected at short retention times from radiochromatograms of plant tissue extracts. The GST gene of the wild-type of Arabidopsis in response to 2,6-DNT was induced by 4.7-fold. However, the uptake rates and the tolerance at different concentrations of 2,6-DNT and TNT were not significantly different between the wild-type and the gst mutant indicating that induction of the GST gene is not related to the detoxification of 2,6-DNT.

Isotopomer measurement techniques in metabolic flux analysis I: nuclear magnetic resonance.

Two-dimensional [(1)H, (13)C] heteronuclear single quantum correlation (HSQC) spectroscopy nuclear magnetic resonance (NMR) is a comprehensive tool in metabolic flux analysis using (13)C-labeling experiments. NMR is particularly relevant when extensive isotopomer measurements are required, such as for plant cells and tissues, which contain multiple cellular compartments. Several isotope isomers (isotopomers) can be detected and their distribution extracted quantitatively from a single 2-D HSQC NMR spectrum. For example, 2-D HSQC detects the labeling patterns of adjacent carbon atoms and provides the enrichment of individual carbon atoms of the amino acids and glucosyl and mannosyl units present in hydrolysates of glycosylated protein. The HSQC analysis can quantitatively distinguish differences between the glucosyl units in the starch hydrolysate and a protein hydrolysate of plant biomass: this specifies crucial information about compartmentalization in the plant system. The peak structures obtained from the HSQC experiment show multiplet patterns that are directly related to the isotopomer abundances. These abundances have a nonlinear relationship to the fluxes via isotopomer balancing. Fluxes are obtained from the numerical solution of these balances and a stoichiometric model that includes biomass composition data as well as consumption rates of carbohydrate and nitrogen sources. Herein, we describe the methods for the experimental measurements for flux analysis, i.e., determination of the biomass composition (lipid, protein, soluble sugar, and starch) as well as detailed procedures of acid hydrolysis of protein and starch samples and NMR sample preparation, using soybean embryo culture as the model plant system. Techniques to obtain the relative intensity of 16 amino acids and glucosyl units for protein hydrolysate and the glucosyl units of starch hydrolysate of soybean embryos in 2-D HSQC NMR spectra also are provided.

Metabolic engineering with plants for a sustainable biobased economy.

Plants are bona fide sustainable organisms because they accumulate carbon and synthesize beneficial metabolites from photosynthesis. To meet the challenges to food security and health threatened by increasing population growth and depletion of nonrenewable natural resources, recent metabolic engineering efforts have shifted from single pathways to holistic approaches with multiple genes owing to integration of omics technologies. Successful engineering of plants results in the high yield of biomass components for primary food sources and biofuel feedstocks, pharmaceuticals, and platform chemicals through synthetic biology and systems biology strategies. Further discovery of undefined biosynthesis pathways in plants, integrative analysis of discrete omics data, and diversified process developments for production of platform chemicals are essential to overcome the hurdles for sustainable production of value-added biomolecules from plants.

Multicomponent nanopatterns by directed block copolymer self-assembly.

Complex nanopatterns integrating diverse nanocomponents are crucial requirements for advanced photonics and electronics. Currently, such multicomponent nanopatterns are principally created by colloidal nanoparticle assembly, where large-area processing of highly ordered nanostructures raises significant challenge. We present multicomponent nanopatterns enabled by block copolymer (BCP) self-assembly, which offers device oriented sub-10-nm scale nanopatterns with arbitrary large-area scalability. In this approach, BCP nanopatterns direct the nanoscale lateral ordering of the overlaid second level BCP nanopatterns to create the superimposed multicomponent nanopatterns incorporating nanowires and nanodots. This approach introduces diverse chemical composition of metallic elements including Au, Pt, Fe, Pd, and Co into sub-10-nm scale nanopatterns. As immediate applications of multicomponent nanopatterns, we demonstrate multilevel charge-trap memory device with Pt-Au binary nanodot pattern and synergistic plasmonic properties of Au nanowire-Pt nanodot pattern.

Analysis of gene expression in poplar trees (Populus deltoides x nigra, DN34) exposed to the toxic explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Poplar plants (Populus deltoides x nigra, DN34) growing under hydroponic conditions were exposed to 50 mg L(-1) of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) for 24 h. The expression of genes potentially involved in the metabolism of toxic explosives was analyzed by reverse-transcriptase (RT) real-time PCR. Genes under study were selected by reference to corresponding genes that were previously shown to be upregulated in the model plant Arabidopsis thaliana by exposure to 2,4,6-trinitrotoluene (TNT) (Ekman et al., 2003. Plant Physiol., 133, 1397-1406). The target genes investigated include several genes encoding for enzymes known to be involved in the detoxification of xenobiotic pollutants, such as glutathione S-transferases (GSTs), cytochrome P-450s (CYPs), NADPH-dependent reductases, and peroxidases. Starting from A. thaliana TNT-inducible genes, corresponding Populus sequences were retrieved from the JGI Poplar Genome Project database and were used to design gene-specific primers. 18S ribosomal DNA (rDNA) was used as an internal standard and recorded gene expression levels were normalized by reference to nonexposed plants. In three separate experiments, five genes were found to be significantly amplified in leaf tissues by exposure to RDX, including GST (9.7 fold), CYP (1.6 fold), reductases (1.6-1.7 fold), and peroxidase (1.7 fold). In root tissues, only a single GST gene was found to be significantly amplified by exposure to RDX (2.0 fold). These results show, for the first time, that the exposure of poplar plants to RDX results in the induction of several genes that are potentially involved in explosive detoxification.

Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar.

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.

Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34).

A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides x nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-(14)C]TNT (25 mg liter(-1)) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of (14)CO(2) was recorded from [(14)C]TNT. In addition, the isolated methylotroph was shown to transform [U-(14)C]RDX (20 mg liter(-1)) and [U-(14)C]HMX (2.5 mg liter(-1)) in less than 40 days. After 55 days of incubation, 58.0% of initial [(14)C]RDX and 61.4% of initial [(14)C]HMX were mineralized into (14)CO(2). The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [(14)C]RDX and [(14)C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.