Such pollution is pervasive worldwide because increasing populations and associated economic growth are diminishing available freshwater, thus leading to increased irrigation of farmlands with waste waters.In the initial soil, copper occurs in two morphological forms . One form decorates coarse organic particles that have some recognizable structures from reticular tissue , and the other occurs in the fine clayey matrix in areas that show organic particulate shapes only at high µ-XRF resolution. In the two phytoremediated soils, similar Cu-organic particulate associations, but also, hot spots of Cu grains 5-20 µm in size were observed in the thin-section maps . In the rhizosphere of P. australis, the Cu hot spots exist outside and in roots and specifically in cortical parenchyma, but not in central vascular cylinders from the stele that contain vascular bundles through which micro-nutrients are transported to reproductive and photosynthetic tissues. In contrast, the main roots and rhizome of I. pseudoacorus do not contain detectable Cu grains, but in the surrounding soil Cu grains are aligned, suggesting that they are associated with biological structures. Under an optical microscope filamentous and ramified organic structures,vertical hydroponic garden similar to root hairs or hyphae from endomycorrhizal fungi, are visible in places where the Cu spots were observed by µ-XRF . Fungal forms are more likely because mycorrhizal hyphae typically are anastomosing, whereas root hairs are not. Fungi may also be implicated in the formation of Cu grains in the cortex of P. australis since roots of this plant are known to be colonized by arbuscular endomycorrhizae in contaminated environments .

These hypotheses are consistent with the capacity of mycorrhizae to accumulate metals and with the storage of Cu in secondary feeder roots of the water hyacinth Eichhornia crassipes . The Cu grains have about the same size as the electron-dense Cu granules in cells of E. splendens placed in a CuSO4 solution for 30 days .Organic Cu.While biological activity clearly modified the original distribution of Cu in the rhizospheres, the Cu species could not be identified from the µ-XRF maps but instead were elucidated using EXAFS spectroscopy. All eight µ-EXAFS spectra from areas in the original soil containing the particle morphologies and chemical compositions observed with µ-XRF can be superimposed on the soil’s bulk EXAFS spectrum , indicating that the initial Cu speciation occurred uniformly. If the initial soil contained various assemblages of Cu species that were distributed unevenly, then we might expect that the proportions of species also would have varied among analyzed areas and been detectable by µ-EXAFS spectroscopy ; however, this was not observed. These spectra match those for Cu2+ binding to carboxyl ligands in natural organic matter, as commonly observed . Elemental Cu. In contrast, only the reference spectrum of elemental copper matches the µ-EXAFS spectra of the 12 hot-spot Cu grains, which are statistically invariant . Photo reduction of Cu2+ in the X-ray beam cannot explain the formation of Cu0 because no elemental Cu was detected in the initial soil by powder and µ-EXAFS, and Cu0 was detected in the two phyto remediated soils at 10 K by bulk EXAFS, and all individual spectral scans from the same sample could be superimposed. At 10 K, radiation damage is delayed , and if Cu had been reduced in the beam, the proportion of Cu0 would have increased from scan to scan which was not observed. Spectra for rhizosphere Cu grains and reference metallic copper have the same phase and overall line shape, but they have significant differences in fine structure and amplitude, which provide details about the nature of the Cu grains.

In the soil Cu grains, shoulders at 5.8 and 7.3 Å-1 are weak and the spectral amplitude is reduced by about 35% and attenuated, relative to metallic copper. The decreased amplitude of the EXAFS signal for the Cu grains relative to well-crystallized metallic copper cannot arise from over absorption because the spectra of the grains were recorded in transmission mode and because the amplitude reduction from over absorption would be uniform in R-space, as demonstrated for ZnS and MnO2 particles , which does not occur in this case. Derived radial structure–functions share the four-peak character of Cu metal . However, long-distance pair correlations are progressively diminished in the soil spectra, indicating multiple interatomic distances , reduced coordination numbers from small particles , and/or abundant micro-structural defects, such as grain and twin boundary dislocations or atomic-scale vacancies. Simulation of the data using multiple-scattering ab initio FEFF calculations showed no evidence for structural disorder .Although the rhizosphere Cu grains are not structurally disordered, their CNs are only 65% , 45% , 38% , and 32% of those in the Cu metal spectrum, indicating that structural order is limited in extent. The lower CN values are consistent with small particles having incompletely coordinated surface atoms. If closed-shell packed and monodispersed, these particles would have a minimum size of 10–15 Å assuming a spherical cuboctahedron shape and 15–20 Å assuming a hemispherical shape, as reported for nanoscale platinum particles . If Cu atoms were missing , as reported for Fe metal powders with a first-shell CN reduced by up to 50% of that in metallic Fe, the effective particle size could be as high as ca. 100 Å in diameter . A defective nanoparticle model is appealing because the surface area-to-volume ratio increases with only a lattice-vacancy parameter.

Constitutive nanoparticles in the micrometer-sized Cu grains are joined at particle or so-called grain boundaries, which might contain stabilizing organic molecules.Complexation to oxygen ligands is possible because EXAFS has relatively low sensitivity to low-Z atoms. However, linear combinations of experimental spectra for organically bound Cu2+ and nanometallic Cu show that the fraction of potential organic Cu in the Cu grains is less than 15%, if present at all . Amounts of Organic and Elemental Cu in the Rhizospheres. Composite bulk EXAFS spectra of the two remediated soils and spectra from the two species identified by µ-EXAFS intersect at the same k values, confirming that only two main Cu species exist in both rhizospheres. Fractional amounts of organically bound Cu from the original soil and metallic Cu formed during phytoremediation were estimated by reconstructing the bulk spectra with linear combinations of the two single species spectra. The best fit for the rhizosphere of P. australis was obtained with 75 . X-ray Diffraction. Featureless two-dimensional µ-XRD patterns from eight Cu hot spots confirm that the Cu grains are aggregates of nanoparticles. However,vertical home farming three patterns display a faint continuous diffraction ring at the Bragg angle for the brightest 111 reflection of Cu metal, indicating larger individual particles with a domain size of 130–150 Å . About 25×104 larger particles would be needed to produce these XRD patterns, but they would comprise only about 0.01% of the analyzed volume . Thus, the diffracting Cu hot spots may have sufficient big particles to yield a powder ring, but they are not enough for their 2NN, 3NN, and higher Cu-Cu shells to contribute significantly to the EXAFS signal. Also, the big particles are undetected by µ-EXAFS because EXAFS signal intensity is linearly proportional to the number of atoms whereas XRD intensity is proportional to the number of atoms squared.The rhizospheres were oxidizing as indicated by the presence of iron oxyhydroxide , absence of sulfide minerals, and the fact that P. australis and I. pseudoacorus are typical wetlands plants with aerenchyma that facilitate oxygen flow from leaves to roots . Thermodynamic calculations using compositions of soil solutions collected below the rhizosphere indicate that Cu+ and Cu2+ species should have been dominant . These points along with the occurrences of nanocrystalline Cu0 in plant cortical cells and as stringer morphologies outside the roots together suggest that copper was reduced biotically. Ecosystem ecology of the rhizosphere indicates synergistic or multiple reactions by three types of organisms: plants, endomycorrhizal fungi, and bacteria. Normally, organisms maintain copper homeostasis through cation binding to bio-active molecules such as proteins and peptides.

When bound, the Cu2+/Cu1+ redox couple has elevated half-cell potentials that facilitate reactions in the electron-transport chain. Even though average healthy cell environments are sufficiently reducing , there are enough binding sites to maintain copper in its two oxidized states. Copper is also important in controlling cell-damaging free radicals produced at the end of the electron-transport chain, for example in the superoxide dismutase enzyme Cu-Zn-SOD, which accelerates the disproportionation of superoxide to O2 and hydrogen peroxide. However, unbound copper ions can catalyze the decomposition of hydrogen peroxide to water and more free radical species. To combat toxic copper and free radicals, many organisms overproduce enzymes such as catalase, chelates such as glutathione, and antioxidants . Mineralizationcould also be a defense against toxic copper, but reports of Cu+ and Cu2+ bio-minerals are rare; only copper sulfide in yeast and copper oxalate in lichens and fungi are known. Atacamite 3Cl in worms does not appear to result from a biochemical defense. Bio-mineralization of copper metal may have occurred by a mechanism analogous to processes for metallic nanoparticle synthesis that exploit ligand properties of organic molecules. In these processes, organic molecules are used as templates to control the shape and size of metallic nanoparticles formed by adding strong reductants to bound cations. For copper nanoparticles and nanowires, a milder reductants as corbic acidshas been used. Ascorbic acid, a well-known antioxidant, reduces Cu cations to Cu0 only when the cations are bound to organic substrates such as DNA in the presence of oxygen in the dark or via autocatalysis on Cu metal seeds in the absence of stabilizing organic ligands . As an example of synthetic control, pH dependent conformation of histidine-rich peptides has led to larger nanocrystals of Cu0 at pH 7–10 than at pH 4–6 . Plants produce ascorbic acid for many functions and rhizospheres often contain the breakdown products of ascorbic acid, which facilitates electron transfer during mineral weathering . Plants produce more ascorbic acid when grown in soils contaminated with heavy metals including copper . Fungi, which proliferate over plants and bacteria in metal-contaminated soils, can stabilize excess copper by extracellular cation binding or oxalate precipitation , but mechanisms probably also require enzymes, thiol-rich proteins and peptides, and antioxidants . The formation of electron-dense Cu granules within hyphae of arbuscular mycorrhizal fungi isolated from Cu- and As-contaminated soil suggests that fungi also can produce nanoparticulate copper. Some copper reduction possibly occurred in response to the European heat wave of the summer of 2003 . Elevated expression of heat shock protein HSP90 and metallothionein genes has been observed in hyphae of an arbuscular mycorrhizal fungus in the presence of 2 × 10–5 M CuSO4 in the laboratory . This suggests that a single driving force can trigger a biological defense mechanism that has multiple purposes. Thus, reduction of toxic cations to native elements may increase as rhizosphere biota fight metal stress and stresses imposed by elevated temperatures expected from global warming, bacteria , and algae can transform other more easily reducible metals, including Au, Ag, Se , Hg, and Te, to their elemental states both intra and extracellularly. When mechanisms have been proposed, they typically have involved enzymes; however, ascorbic acid was implicated when Hg2+ was transformed to Hg0 in barley leaves . Theoretically all of these metal cations could be transformed by a reducing agent weaker than ascorbic acid . However, binding appears to stabilize cationic forms in the absence of a sufficiently strong reductant such as ascorbic acid. Processes used in materials synthesis that were developed with biochemical knowledge might yield clues to other possible, but presently unknown, biologically mediated reactions in different organisms.The discovery of nanoparticulate copper metal in phytoremediated soil may shed light on the occurrence of copper in peats.However, swamps by definition are more oxidizing with neutral to alkaline pHs, and they may be ideal sites for biotic formation of metallic Cu nanoparticles. For example, in swamp peats near Sackville, New Brunswick, Canada, copper species unidentifiable by XRD were dissolved only with corrosive perchloric acid , suggesting they may have been nanoparticulate metal formed by active root systems of swamp plants. If swamp peats evolve to bog peats the Cu reduction mechanism could convert to autocatalysis on the initial nanocrystals . The addition of peats that either act as templating substrates or contain nanoparticulate copper could enhance the effectiveness of using wetlands plants for phytoremediation.