Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.

Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.

Custom software entitled Plant Metabolite Annotation Toolbox (PlantMAT) has been developed to address the number one grand challenge in metabolomics, which is the large-scale and confident identification of metabolites. PlantMAT uses informed phytochemical knowledge for the prediction of plant natural products such as saponins and glycosylated flavonoids through combinatorial enumeration of aglycone, glycosyl, and acyl subunits. Many of the predicted structures have yet to be characterized and are absent from traditional chemical databases, but have a higher probability of being present in planta. PlantMAT allows users to operate an automated and streamlined workflow for metabolite annotation from a user-friendly interface within Microsoft Excel, a familiar, easily accessed program for chemists and biologists. The usefulness of PlantMAT is exemplified using ultrahigh-performance liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC ESI-QTOF-MS/MS) metabolite profiling data of saponins and glycosylated flavonoids from the model legume Medicago truncatula. The results demonstrate PlantMAT substantially increases the chemical/metabolic space of traditional chemical databases. Ten of the PlantMAT-predicted identifications were validated and confirmed through the isolation of the compounds using ultrahigh-performance liquid chromatography mass spectrometry solid-phase extraction (UHPLC MS SPE) followed by de novo structural elucidation using 1D/2D nuclear magnetic resonance (NMR). It is further demonstrated that PlantMAT enables the dereplication of previously identified metabolites and is also a powerful tool for the discovery of structurally novel metabolites.

Analytical artifacts attributed to the bromination of toluene, xylenes, and trimethylbenzenes were found during the heated headspace gas chromatography/mass spectrometry (GC/MS) analysis of aqueous samples. The aqueous samples were produced from Fenton-like chemical oxidation reactions and contained aromatic compounds, hydrogen peroxide (H2O2), and ferric sulfate. Prior to GC/MS headspace analysis, the samples were acidified (pH<2), and sodium chloride was amended to the headspace vial as a matrix modifier. The brominated artifacts were generated during heated headspace analysis. Further, when samples were spiked with a mixture of volatile chlorinated and aromatic compounds (50 µg/L), poor spike recoveries of toluene and xylenes occurred, and in some cases complete loss of trimethylbenzenes and naphthalene resulted. Where poor recovery of aromatic spike compounds occurred, brominated aromatic compounds were found. The only significant source of bromine in the reaction scheme is the bromide typically present (<0.01% w/w) in the sodium chloride amended to the samples. Conversely, brominated artifacts were absent when a buffered salt mixture composed of sodium chloride and potassium phosphate dibasic/monobasic was used as a matrix modifier and raised the sample pH (pH~6). This indicated that the brominated artifacts resulted from the reaction of the aromatic compounds with BrCl, which was formed by the reaction of H2O2, chloride, and bromide under acidic conditions. An alternative matrix modifier salt is recommended that prevents the bromination reaction and avoids these deleterious effects on sample integrity during headspace analysis.

Microcosms were constructed with sediment from beneath a landfill that received waste containing PFOA (perfluorooctanoic acid) and PFOS (perfluorooctane sulfonate). The microcosms were amended with PFOA and PFOS, and sampled after 91, 210, 343, 463, 574, and 740 d of incubation. After 740 d, selected microcosms were extracted to determine the mass of PFOA and PFOS remaining. There was no evidence for degradation of PFOA or PFOS. Over time, the aqueous concentrations of PFOA and PFOS increased in the microcosms, indicating that PFOA and PFOS that had originally sorbed to the sediment was desorbing. At the beginning of the experiment, the adsorption coefficient, Kd, averaged 0.27 L/kg for PFOA and 1.2 L/kg for PFOS. After 740 d of incubation, sorption of PFOA was not detectable and the Kd of PFOS was undetectable in two microcosms and was 0.08 L/kg in a third microcosm. During incubation, the pH of the pore water in the microcosms increased from pH 7.2 to pH ranging from 8.1 to 8.8. The zeta potential of the sediment decreased with increasing pH. These observations suggest that the sorption of PFOA and PFOS at near neutral pH was controlled by the electrostatic sorption on ferric oxide minerals, and not by the sorption to organic carbon. Accurate predictions of PFOA and PFOS mobility in ground water should be based on empirical estimates of sorption using affected aquifer sediment.

In Fenton-driven oxidation treatment systems, reaction intermediates derived from parent compounds can play a significant role in the overall treatment process. Fenton-like reactions in the presence of toluene or benzene, involved a transformation mechanism that was highly efficient relative to the conventional Fenton-driven mechanism. A delay in hydrogen peroxide (H2O2) reaction occurred until the complete or near-complete transformation of toluene or benzene and involved the simultaneous reaction of dissolved oxygen. This highly efficient transformation mechanism is initiated by Fenton-like reactions, and therefore dependent on conventional Fenton-like parameters. Results indicated that several potential parameters and mechanisms did not play a significant role in the transformation mechanism including electron shuttles, Fe chelates, high valent oxo-iron complexes, anionic interferences in H2O2 reaction, and H2O2 formation. The Fenton-like initiation, formation, and propagation of a reaction intermediate species capable of transforming toluene, while simultaneously inhibiting H2O2 reaction is the most viable mechanism.

A method was developed to determine the vapor concentration of tetrachloroethylene (PCE) at and below its equilibrium vapor-phase concentration, 168 000 microg/L (25 degrees C). Vapor samples were drawn by vacuum into a six-port sampling valve and injected through a jet separator into an ion trap mass spectrometer (MS). This on-line MS can continuously sample a vapor stream and provide vapor concentrations every 30 s. Calibration of the instrument was done by creating a saturated stream of PCE vapor, sampling the vapor with the on-line MS and with thermal desorption tubes, and correlating the peak area response from the MS with the vapor concentration determined by automated thermal desorption gas chromatography mass spectrometry. Dilution of the saturated stream provided lower concentrations of PCE vapor. The method was developed to monitor the vapor concentration of PCE that was sparged from a two-dimensional flow chamber and for determination of the total PCE mass removed during each sparge event. The method has potential application for analysis of gas-phase tracers.

At room temperature (20° ± 3°C), purge and trap samplers provide poor sensitivity for analysis of the fuel oxygenates that are alcohols, such as tertiary butyl alcohol (TBA). Because alcohols are miscible or highly soluble in water, they are not efficiently transferred to a gas chromatograph for analysis. To improve the efficiency of transfer, the water in a purge and trap sampler can be heated. Alternatively, the sensitivity for TBA can be improved by preparing the sample in a heated static headspace sampler. In a heated water sample, the acid used as a preservative may cause chemical hydrolysis of methyl tertiary butyl ether (MTBE) to produce TBA. This effect is well illustrated in this paper using data collected by the U.S. Environmental Protection Agency Office of Research and Development on a plume of MTBE in California. Samples were analyzed using a static head‐space sampler heated to 80°C. The ground water samples were preserved in the field with HCl to a pH ≤ 2. The extent of MTBE hydrolysis to TBA during sample analysis varied from 19% to 87%; the average extent of hydrolysis was 59%. To confirm and document the importance of acid hydrolysis of MTBE at higher temperatures during sample preparation, the rate of hydrolysis of MTBE was measured at 80°C. At pH = 1, the rate of hydrolysis was 1.22/h, while the rate at pH = 2 was 0.15/h. Acid hydrolysis of MTBE during sample preparation in a heated headspace sampler can be avoided by preserving the sample with 1% (w/w) trisodium phosphate dodecahydrate (TSP) instead of using HCl, or by neutralizing the acid before analysis. In the presence of an acclimated microbial culture, TSP prevented biodegradation of MTBE, as well as benzene, toluene, ethylbenzene, and xylene compounds, in ground water at room temperature for 66 d. However, in a spike recovery experiment, TSP caused based catalyzed hydrolysis of bromomethane. It is not appropriate as a universal preservative.

The determination of fuel ether oxygenates in groundwater was found to be problematic when samples are preserved at pH < 2 and then analyzed using heated headspace sampling. Acid catalyzed the hydrolysis of tert-amyl methyl ether, ethyl tert-butyl ether, and methyl tert-butyl ether during headspace sampling when aqueous samples were heated at 80 degrees C, a typical temperature used for heated headspace sampling. Hydrochloric acid at pH 2 did not cause hydrolysis of oxygenate ethers in samples stored for 28 d at 4 degrees C. When trisodium phosphate was used to preserve the sample or to adjust the pH of samples preserved with acid before headspace sampling, the recovery of spiked ethers was excellent. The heated headspace method was also applicable for the determination of other fuel oxygenates including ethanol, tert-butyl alcohol (TBA), tert-amyl alcohol (TAA), isopropyl alcohol (IPA), acetone, and monoaromatic compounds found in gasoline including benzene, toluene, ethylbenzene, xylenes, and trimethylbenzenes. The method detection limits range from 0.1 to 0.2 microg/L for the ethers and aromatics. For alcohols and acetone, the method detection limits were 0.8 microg/L for TBA, 18 microg/L for ethanol, 1.2 microg/L for TAA, 5.5 microg/L for IPA, and 3.3 microg/L for acetone. The heated headspace method yielded accurate results for ether oxygenates in samples containing a wide range of gasoline concentrations (2500-100000 microg/L).

A method was developed for the confirmed identification and quantitation of 17β-estradiol, estrone, 17α-ethynylestradiol and 16α-hydroxy-17β-estradiol (estriol) in ground water and swine lagoon samples. Centrifuged and filtered samples were extracted using solid-phase extraction (SPE), and extracts were derivatized using pentafluorobenzyl bromide (PFBBR) and N-trimethylsilylimidazole (TMSI). Analysis was done using negative ion chemical ionization (NICI) gas chromatography–mass spectrometry–mass spectrometry (GC–MS–MS). Deuterated analogs of each of the estrogens were used as isotope dilution standards (IDS) and were added to the samples before extraction. A limit of quantitation of 1ng/l in ground water was obtained using 500ml of ground water sample, 1.0ml of extract volume and the lowest calibration standard of 0.5pg/μl. For a 25ml swine lagoon sample, the limit of quantitation was 40ng/l. The average recovery of the four estrogens spiked into 500ml of distilled water and ground water samples (n=16) at 2ng/l was 103% (S.D. 14%). For 25ml of swine lagoon samples spiked at 500, 1000 and 10,000ng/l, the average recovery for the four estrogens was 103% (S.D. 15%). The method detection limits (MDLs) of the four estrogens spiked at 2ng/l in a 500ml of ground water sample ranged from 0.2 to 0.6ng/l. In swine lagoon samples from three different types of swine operations, estrone was found at levels up to 25,000ng/l, followed by estriol and estradiol up to levels at 10,000 and 3000ng/l, respectively. It was found that pretreatment of swine lagoon samples with formaldehyde was necessary to prevent conversion of estradiol to estrone.

Recently, two papers reported the use of solid-phase microextraction (SPME) with poly(dimethylsiloxane)(PDMS)/Carboxen fibers to determine trace levels of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (tBA) in water. Attempts were made to apply this technique to the analysis of water samples containing high levels of benzene, toluene, ethylbenzene, xylenes, and trimethylbenzenes (BTEXsTMBs) as would be expected at leaking underground storage tank sites. It was found that when the sample contained total aromatic compounds above 1 ppm, the response of the internal standards, deuterated MTBE and tBA, dropped by more than 65%. As this decrease in internal standard peak area was unacceptable, a static headspace method was used instead. This headspace method was used successfully to analyze groundwater from 670 monitoring wells at 74 service stations located in the northeast United States. In these monitoring wells, 30% of the samples contained total BTEXsTMBs above 1 ppm. If the SPME method was used to analyze these samples, dilution of more than 200 samples would be required to minimize the adverse matrix effect that high aromatic content had on the internal standard peak area.

A ground-water treatment process is described in which contaminants are adsorbed onto granulated activated carbon (GAC) containing fixed iron oxide. Hydrogen peroxide (H2O2) is amended to the GAC suspension and reacts with the iron, forming hydroxyl radicals ((OH)-O-.). The radicals react with and oxidize sorbed and soluble contaminants regenerating the carbon surface. Laboratory results are presented in which 2-chloro-phenol (2CP) was first adsorbed to GAC and subsequently oxidized via the Fenton-driven mechanism. Transformation of 2CP was indicated by the formation of carboxylic acids and Cl(-)release. The treatment efficiency of 2CP, defined as the molar ratio of Cl(-)released to H2O2 consumed, increased with increasing amounts of iron oxide and 2CP on the GAG. The extent of 2CP oxidation increased with H2O2 concentration. Lower treatment efficiency was evident at the highest H2O2 concentration utilized (2.1 M) and was attributed to increased (OH)-O-. scavenging by H2O2. Aggressive oxidation procedures used in sequential adsorption/oxidation cycles did not alter the GAC surface to a degree that significantly interfered with subsequent 2CP adsorption reactions. Although process feasibility has not yet been established beyond bench-scale, experimental results illustrate the potential utility of the adsorption/oxidation process in aboveground systems or permeable reactive barriers for the treatment of contaminated ground water.

A spill of JP-4 jet fuel at the U.S. Coast Guard Air Station in Traverse City, Michigan, contaminated a water-table aquifer. An infiltration gallery (30 ft x 30 ft) was installed above a section of the aquifer containing 700 gal JP-4. Purge wells recirculated three million gallons of ground water per week through the infiltration gallery at a rate designed to raise the water table above the contaminated interval. Ground water containing ambient concentrations of oxygen and nitrate was first recirculated for 40 days. Concentrations of benzene in monitoring wells beneath the infiltration gallery were reduced from 760 to < 1-mu-g/l. Concentrations of toluene, ethylbenzene, m,p-xylene, and o-xylene were reduced from 4500 to 17, 840 to 44, 2600 to 490, and 1400 to 260-mu-g/l, respectively. Average core concentrations of benzene, toluene, ethylbenzene, m,p-xylene, and o-xylene were reduced from 0.84 to 0.032, 33 to 0.13, 18 to 0.36, 58 to 7.4, and 26 to 3.2 mg/kg, respectively. Ground water amended with nitrate (10 mg/l nitrate-nitrogen) and nutrients was then recirculated for 76 days. Final core concentrations of benzene, toluene, ethylbenzene, m,p-xylene, and o-xylene were 0.017, 0.036, 0.019, 0.059, and 0.27 mg/kg, respectively. Final aqueous concentrations were < 1-mu-g/l for benzene and toluene, 6-mu-g/l for ethylbenzene, and 20 to 40-mu-g/l for the xylene isomers, in good agreement with predicted values based on residual fuel content and partitioning theory. Although alkylbenzene concentrations have been substatially reduced, the test plot is still contaminated with the weathered fuel. Based on stoichiometry, approximately 10 times more nitrate was consumed than could be accounted for by BTX degradation alone, indicating that other compounds were also degraded under denitrifying conditions.