Investigating the Potential of Fusarium solani and Phanerochaete chrysosporium in the Removal of 2,4,6-TNT.

Past and recent applications of 2,4,6-trinitrotoluene (TNT) in military and civilian industries have led to contamination of soil and marine ecosystems. Among various TNT remediation techniques, biological remediation is widely accepted for its sustainability, low cost, and scalable applications. This study was designed to isolate a fungus strain from a TNT-contaminated soil to investigate its tolerance to and potential for removal of TNT. Thus, a soil column with a history of periodic TNT amendment was used to isolate dominant strains of fungi Fusarium solani isolate, which is not commonly reported for TNT mineralization and was found predominant in the subsurface layer of the TNT-amended soil. F. solani was investigated for TNT concentration tolerance at 30, 70, and 100 mg/L on agar plates and for TNT removal in liquid cultures at the same given concentrations. F. solani activity was compared with that of a reference soil-born fungus that has been intensively studied for TNT removal (Phanerochaete chrysosporium) obtained from the American Type Culture Collection. On agar media, F. solani showed a larger colony diameter than P. chrysosporium at similar TNT concentrations, indicating its high potential to tolerate toxic levels of TNT as found in contaminated sites. In the liquid culture medium, F. solani was able to significantly produce higher biomass than P. chrysosporium in all TNT concentrations. The TNT removal percentage from the liquid culture at the highest TNT concentration of 100 mg/L reached about 85% with F. solani, while P. chrysosporium was no better than 25% at the end of an 84-h incubation period. Results indicate a significant potential of using F. solani in the bioremediation of polluted TNT soils that overcome the high concentration barrier in the field. However, further investigation is needed to identify enzymatic potential and the most effective applications and possible limitations of this method on a large scale.

Phytostabilization of a contaminated military site using Miscanthus and soil amendments.

Military activities can contaminate productive land with potentially toxic substances. The most common trace metal contaminant on military bases is lead (Pb). A field experiment was begun in 2016 at Fort Riley, KS, in an area with total soil Pb concentrations ranging from 900 to 1,500 mg kg-1 and near-neutral pH. The main objectives were to test the potential of Miscanthus sp. for phytostabilization of the site and to evaluate the effects of soil amendments on Miscanthus growth, soil-plant Pb transfer, bioaccessibility of soil Pb, and soil health. The experimental design was a randomized complete block, with five treatments and four replications. Treatments were (a) existing vegetation; (b) Miscanthus planted in untilled soil, no amendments; (c) Miscanthus planted in tilled soil; (d) Miscanthus planted in tilled soil amended with inorganic P (triple superphosphate applied at 5:3 Pb:P); and (e) Miscanthus planted in tilled soil amended with organic P (Class B biosolids applied at 45 Mg ha-1 ). Tilling and soil amendments increased dry matter yields only in the establishment year. Total Pb uptake, plant tissue Pb concentration, and soil Pb bioaccessibility were significantly less in the Miscanthus plots amended with biosolids than the Miscanthus plots with no added P across all 3 yr. Enzyme activities, organic carbon, and microbial biomass were also greater in biosolids-treated plots. Results show that planting-time addition of soil amendments to Pb-contaminated soil supported Miscanthus establishment, stabilized and reduced bioaccessibility of soil Pb, reduced concentration and uptake of Pb by Miscanthus, and enhanced soil health parameters.

Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

Phytoremediation in education: textile dye teaching experiments.

Phytoremediation, the use of plants to clean up contaminated soil and water, has a wide range of applications and advantages, and can be extended to scientific education. Phytoremediation of textile dyes can be used as a scientific experiment or demonstration in teaching laboratories of middle school, high school and college students. In the experiments that we developed, students were involved in a hands-on activity where they were able to learn about phytoremediation concepts. Experiments were set up with 20-40 mg L(-1) dye solutions of different colors. Students can be involved in the set up process and may be involved in the experimental design. In its simplest forms, they use two-week-old sunflower seedlings and place them into a test tube of known volume of dye solution. Color change and/or dye disappearance can be monitored by visual comparison or with a spectrophotometer. Intensity and extent of the lab work depends on student's educational level, and time constraints. Among the many dyes tested, Evan's Blue proved to be the most readily decolorized azo dye. Results could be observed within 1-2 hours. From our experience, dye phytoremediation experiments are suitable and easy to understand by both college and middle school students. These experiments help visual learners, as students compare the color of the dye solution before and after the plant application. In general, simple phytoremediation experiments of this kind can be introduced in many classes including biology, biochemistry and ecological engineering. This paper presents success stories of teaching phytoremediation to middle school and college students.

Infrared imaging of sunflower and maize root anatomy.

Synchrotron radiation infrared microspectroscopy (SR-IMS) permits the direct analysis of plant cell-wall architecture at the cellular level in situ, combining spatially localized information and chemical information from IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers, such as cellulose, lignin, and proteins, in the root tissue of hydroponically grown sunflower and maize plants. Principal components analysis (PCA) was employed to reveal the major spectral variance between maize and sunflower plant tissues. The use of PCA showed distinct separation of maize and sunflower samples using the IR spectra of the epidermis and xylem. The infrared band at 1635 cm(-1), representing hydrocinnamic acid in (H type) lignin, provided a conclusive means of distinguishing between maize and sunflower plant tissues.

Elicitor signal transduction leading to production of plant secondary metabolites.

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and other industrial materials. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Understanding signal transduction paths underlying elicitor-induced production of secondary metabolites is important for optimizing their commercial production. This paper summarizes progress made on several aspects of elicitor signal transduction leading to production of plant secondary metabolites, including: elicitor signal perception by various receptors of plants; avirulence determinants and corresponding plant R proteins; heterotrimeric and small GTP binding proteins; ion fluxes, especially Ca2+ influx, and Ca2+ signaling; medium alkalinization and cytoplasmic acidification; oxidative burst and reactive oxygen species; inositol trisphosphates and cyclic nucleotides (cAMP and cGMP); salicylic acid and nitric oxide; jasmonate, ethylene, and abscisic acid signaling; oxylipin signals such as allene oxide synthase-dependent jasmonate and hydroperoxide lyase-dependent C12 and C6 volatiles; as well as other lipid messengers such as lysophosphatidylcholine, phosphatidic acid, and diacylglycerol. All these signal components are employed directly or indirectly by elicitors for induction of plant secondary metabolite accumulation. Cross-talk between different signaling pathways is very common in plant defense response, thus the cross-talk amongst these signaling pathways, such as elicitor and jasmonate, jasmonate and ethylene, and each of these with reactive oxygen species, is discussed separately. This review also highlights the integration of multiple signaling pathways into or by transcription factors, as well as the linkage of the above signal components in elicitor signaling network through protein phosphorylation and dephosphorylation. Some perspectives on elicitor signal transduction and plant secondary metabolism at the transcriptome and metabolome levels are also presented.

Temperature and pH effects on plant uptake of benzotriazoles by sunflowers in hydroponic culture.

This article describes a systematic approach to understanding the effect of environmental variables on plant uptake (phyto-uptake) of organic contaminants. Uptake (and possibly phytotransformation) of xenobiotics is a complex process that may differ from nutrient uptake. A specific group of xenobiotics (benzotriazoles) were studied using sunflowers grown hydroponically with changes of environmental conditions including solution volume, temperature, pH, and mixing. The response of plants to these stimuli was evaluated and compared using physiological changes (biomass production and water uptake) and estimated uptake rates (influx into plants), which define the uptake characteristics for the xenobiotic. Stirring of the hydroponic solution had a significant impact on plant growth and water uptake. Plants were healthier, probably because of a combination of factors such as improved aeration and increase in temperature. Uptake and possibly phytotransformation of benzotriazoles was increased accordingly. Experiments at different temperatures allowed us to estimate an activation energy for the reaction leading to triazole disappearance from the solution. The estimated activation energy was 43 kJ/mol, which indicates that the uptake process is kinetically limited. Culturing plants in triazole-amended hydroponic solutions at different pH values did not strongly affect the biomass production, water uptake, and benzotriazole uptake characteristics. The sunflowers showed an unexpected capacity to buffer the solution pH.

Phytotransformation of benzotriazoles.

Plant roots interact with organic pollutants and some of these contaminants can be phytotransformed. Root uptake of 1-H-benzotriazole and its derivatives, tolyltriazole, 5-methyl benzotriazole, and 1-hydroxy benzotriazole was studied. At levels below the toxic threshold of about 100 mg/L, triazoles appear to be incorporated into plant tissue. Their concentration in the aqueous phase of the culture decreases with time and they cannot be extracted from the plant material using methanol. Hydroponic studies with sunflowers (Helianthus annuus) were used to investigate the behavior of the solution concentration versus time and to determine kinetic parameters for plant uptake of triazoles. Plants actively take up the triazoles at a rate greater than predicted by transpiration stream-concentration factor and plant-water uptake. Analyses of the data for phytotransformation rate versus concentration were performed to establish the kinetic model for the removal process. Except for 1-hydroxy-benzotriazole, triazole disappearance in plant systems followed the Michaelis-Menten kinetic model (commonly found for enzyme-catalyzed reactions) better than a first order rate model. However, the fit for the first order model was improved when normalizing to the plant fresh weight, which was assumed to be an approximate measure of the changing root surface area. Experiments with other plant species are in progress.