Inoculation of soil or seeds with plant growth promoting bacteria ameliorates metal toxicity to plants by changing metal speciation in plant tissues but the exact location of these changes remains unknown. Knowing where the changes occur is a critical first step to establish whether metal speciation changes are driven by microbial metabolism or by plant responses. Since bacteria concentrate in the rhizosphere, we hypothesised steep changes in metal speciation across the rhizosphere. We tested this by comparing speciation of zinc (Zn) in roots of Brassica juncea plants grown in soil contaminated with 600 mg kg⁻¹ of Zn with that of bulk and rhizospheric soil using synchrotron X-ray absorption spectroscopy (XAS). Seeds were either uninoculated or inoculated with Rhizobium leguminosarum bv. trifolii and Zn was supplied in the form of sulfide (ZnS nanoparticles) and sulfate (ZnSO₄). Consistent with previous studies, Zn toxicity, as assessed by plant growth parameters, was alleviated in B. juncea inoculated with Rhizobium leguminosarum. XAS results showed that in both ZnS and ZnSO₄ treatments, the most significant changes in speciation occurred between the rhizosphere and the root, and involved an increase in the proportion of organic acids and thiol complexes. In ZnS treatments, Zn phytate and Zn citrate were the dominant organic acid complexes, whilst Zn histidine also appeared in roots exposed to ZnSO₄. Inoculation with bacteria was associated with the appearance of Zn cysteine and Zn formate in roots, suggesting that these two forms are driven by bacterial metabolism. In contrast, Zn complexation with phytate, citrate and histidine is attributed to plant responses, perhaps in the form of exudates, some with long range influence into the bulk soil, leading to shallower speciation gradients.

The phytoextraction potential of plants for removing heavy metals from polluted soils is determined by their capacity to store contaminants in aboveground organs and complex them safely. In this study, the metal compartmentation, elemental composition of zinc deposits and zinc complexation within leaves from poplars grown on soil with mixed metal contamination was analysed combining several histochemical and microanalytical approaches. Zinc was the only heavy metal detected and was stored in several organelles in the form of globoid deposits showing β-metachromasy. It was associated to oxygen anions and different cations, noteworthy phosphorous. The deposit structure, elemental composition and element ratios indicated that zinc was chelated by phytic acid ligands. Maturation processes in vacuolar vs. cytoplasmic deposits were suggested by differences in size and amounts of complexed zinc. Hence, zinc complexation by phytate contributed to metal detoxification and accumulation in foliage but could not prevent toxicity reactions therein.

While phosphate (P) inhibits arsenic (As) uptake by plants, phytate increases As uptake by As-hyperaccumulator Pteris vittata. Here we tried to understand the underling mechanisms by investigating the roles of phytate in soil As desorption, P transport in P. vittata, short-term As uptake, and plant growth and As accumulation from soils. Sterile soil was used to exclude microbial degradation on phytate. Results showed that inorganic P released 3.3-fold more As than that of phytate from soil. However, P. vittata accumulated 2–2.5 fold more As from soils with phytate than that in control and P treatment. In addition, different from P suppression on As uptake, solution uptake experiment showed that As uptake in phytate treatment was comparable to that of control under 0.1–7.5 μM As after 1–24 h. Moreover, responding to phytate, P. vittata P transporter PvPht1;3 increased by 3-fold while PvPht1;1 decreased by 65%. The data suggested that phytate upregulated PvPht1;3, thereby contributing to As uptake in P. vittata. Our results showed that, though with lower As release from soil compared to P, phytate induced more As uptake and better growth in P. vittata by upregulating P transporters.

Phytate is abundant in soils, which is stable and unavailable for plant uptake. However, it occurs in root exudates of As-hyperaccumulator Pteris vittata (PV). To elucidate its effect on As uptake and growth, P. vittata was examined on agar media (63 μM P) containing 50 μM As and/or 50 or 500 μM phytate with non As-hyperaccumulator Pteris ensiformis (PE) as a congeneric control. Phytate induced efficient As and P uptake, and enhanced growth in PV, but had little effects on PE. The As concentrations in PV fronds and roots were 157 and 31 mg kg−1 in As50+phytate50, 2.2- and 3.1-fold that of As50 treatment. Phosphorus uptake by PV was reduced by 27% in As treatment than the control (P vs. P + As) but increased by 73% comparing phytate500 to phytate500+As, indicating that PV effectively took up P from phytate. Neither As nor phytate affected Fe accumulation in PV, but phytate reduced root Fe concentration in PE (46–56%). As such, the increased As and P and the unsuppressed Fe uptake in PV probably promoted PV growth. Thus, supplying phytate to As-contaminated soils may promote As uptake and growth in PV and its phytoremediation ability.

Phosphorus (P) is limiting nutrient in many soils, and P availability may often depend on iron (Fe) speciation. Colloidal iron phosphate (FePO₄cₒₗₗ) is potentially present in soils, and we tested the hypothesis that phytate exudation by Pteris vittata might dissolve FePO₄cₒₗₗ by growing the plant in nutrient solution to which FePO₄cₒₗₗ was added. The omission of P and Fe increased phytate exudation by P. vittata from 434 to 2136 mg kg⁻¹ as the FePO₄cₒₗₗ concentration increased from 0 to 300 mM. The total P in P. vittata tissue increased from 2880 to 8280 mg kg⁻¹, and the corresponding increases in the trichloroacetic acid (TCA) extractable P fractions were inorganic P (860–5100 mg kg⁻¹), soluble organic P (250–870 mg kg⁻¹), and insoluble organic P (160–2030 mg kg⁻¹). That is, FePO₄-solubilizing activity was positive correlated with TP, TCA P fractions in P. vittata, TP in growth media, and root exudates. This study shows that phytate exudation dissolved FePO₄cₒₗₗ due to the chelation effect of phytic acid on Fe; however, the wider question of whether phytic acid excretion was prompted by deprivation of P, Fe, or both remains to be answered.

The mechanisms by which complex products released from the organic matrix of cattle manure impact phosphorus (P) behavior and transport are largely undefined. Effects of a dairy slurry isolate on sorption characteristics of three benchmark soils and the breakthrough of phytate-P were determined in short soil columns of Mattapex loam (fine-silty, mixed, active, mesic Aquic Hapludults) under saturated flow conditions. The manure liquid isolate was obtained after a 7-day incubation of reconstituted dairy manure (1.6:1, feces-to-urine) at 37 °C and centrifugation at 16,000×g. The liquid isolate, at dilutions of 20:1 to 4:1 water-to-isolate, decreased soil sorption of phytate-P, with reduction in logₑ K averaging 30%. Whether the influent contained artificial rainwater or the manure isolate at a water-to-isolate ratio of 20:1, P retention and breakthrough curves were differentially impacted. Only inorganic phosphate-P was eluted in a multiple-stage process, and breakthrough occurred after 16 pore volumes of rainwater. Both inorganic- and enzyme-labile P (TBIOP) appeared in the effluent when either a dilute solution of 0.05 M EDTA (ethylenediamine-N, N, N′, N′-tetraacetate) or one containing 5% of manure liquid isolate was used as influent. The polydentate ligand-like substances reduced (i) the soil’s affinity for phytate and (ii) the hydrolysis rate in soil, allowing phytate to be eluted. Therefore, dissolved components of the manure matrix played critical roles in controlling transport and dispersion of phytate-derived P forms in soil and may hold the key to the understanding of biogeochemical bases of persistent effects of legacy P in agricultural watersheds.

Heat- and pH-stable phytase efficiently hydrolyzes phytic acid. In this research, heat- and pH-stable mutant phytases, T⁸³R, L²⁸⁷R, and T⁸³R/L²⁸⁷R were generated by site-directed mutagenesis from Yersinia intermedia. After the induction and expression of recombinant wild-type and mutant phytases in E. coli BL21, the enzymes were purified using nickel sepharose affinity chromatography, and characterized kinetically and thermodynamically using spectroscopy methods. The mutants showed optimum activity at pH 5.15 and 55–61 °C. The catalytic efficiencies of T⁸³R, L²⁸⁷R, T⁸³R/L²⁸⁷R, and wild-type phytases were calculated to be 2941, 29346, 4906, and 6917 mmol/L⁻¹s⁻¹, respectively. Moreover, after the incubation of T⁸³R, L²⁸⁷R, wild-type, and T⁸³R/ L²⁸⁷R phytases at 100 °C for 1 h, the enzymes retained 22, 5, 4, and 2% of their initial activities, respectively. In addition, T⁸³R, T⁸³R/L²⁸⁷R, L²⁸⁷R, and wild-type phytases retained 82, 44, 16 as well as 11% of their initial activities after 1 h at pH 5.15, respectively. Among these mutants, T⁸³R mutant showed 18% increase in thermal stability, 71% increase in pH stability, and +0.103 KJ/mole increase in ΔΔG, while the catalytic efficiency and ΔΔG value of L²⁸⁷R mutant increased by 4 times and +0.0903 KJ/mole, respectively. Thus, the mutants have the potential to be used in feed industries to increase the bioavailability of minerals while decreasing soil and water pollution.

Calcareous soil, high pH, and low organic matter are the major factors that limit iron (Fe) availability to rice crop. The present study was planned with the aim to biofortified rice grain with Fe, by integrated use of chemical and organic amendments in pH-manipulated calcareous soil. The soil pH was reduced (pHL₂) by using elemental sulfur (S) at the rate of 0.25 % (w/w). The organic amendments, biochar (BC) and poultry manure (PM) [1 % (w/w)], along with ferrous sulfate at the rate of 7.5 mg kg⁻¹ soil were used. The incorporation of Fe with BC in soil at pHL₂ significantly improved plant biomass, photosynthetic rate, and paddy yield up to 99, 97, and 36 %, respectively, compared to control. A significant increase in grain Fe (190 %), protein (58 %), and ferritin (400 %) contents was observed while anti-nutrients, i.e., polyphenols (37 %) and phytate (21 %) were significantly decreased by the addition of Fe and BC in soil at pHL₂ relative to control. Among the organic amendments, PM significantly increased Cd, Pb, Ni, and Cr concentrations in rice grain relative to control but their concentration values were below as compared to the toxic limits of hazard quotients and hazard index (HQ and HI). Hence, this study implies that Fe applied with BC in the soil at pHL₂ can be considered as an effective strategy to augment Fe bioavailability and to reduce non-essential heavy metal accumulation in rice grain.

Sorption of organic phosphates–myo-inositol hexakisphosphate (IHP) and glycerol phosphate (GP) and its effects on the early stage of hematite aggregation kinetics were investigated at different pH and electrolyte composition. KH₂PO₄ (KP) was taken as an inorganic P source for comparison. Results indicated that for all types of P, the sorption amounts decreased with increasing solution pH. Sorption amount of IHP was almost two times that of KP, while those of GP and KP were close. Both organic P and inorganic P interacted with hematite via ligand exchange through their phosphate groups, which conveyed negative charges to mineral surface and significantly decreased the zeta potential of hematite. In Na⁺ solution, critical coagulation concentrations (CCCs) of hematite suspensions increased with increasing P concentration and followed the order of KP < GP < IHP at pH 5.5. Compared with KP, the organic P could more effectively stabilize the hematite suspension not only through increasing the negative charges and electrostatic repulsive force, but also through steric repulsion between P-sorbed hematite nanoparticles. When the pH was increased from 5.5 to 10.0, the CCCs of the hematite suspensions with GP and IHP decreased mainly because of the great reductions in organic P sorption amounts and consequent decreases in electrostatic and steric repulsive forces. However, enhanced aggregation was observed in the presence of IHP at pH 4.5 and above in low Ca²⁺ solutions. The precipitation of calcium phytate formed net-like structure, which served as bridges to bind hematite nanoparticles and resulted in enhanced aggregation. These results have important implications for assessing the fate and transport of organic P and hematite nanoparticles in soil and aquatic environments.

Rice (Oryza sativa L.) is one of the main staple food crops which is inherently low in micronutrients, especially iron (Fe), and can lead to severe Fe deficiency in populations having higher consumption of rice. Soils polluted with nickel (Ni) can cause toxicity to rice and decreased Fe uptake by rice plants. We investigated the potential role of biochar (BC) and gravel sludge (GS), alone and in combination, for in situ immobilization of Ni in an industrially Ni-contaminated soil at original and sulfur-amended altered soil pH. Our further aim was to increase Fe bioavailability to rice plants by the exogenous application of ferrous sulfate to the Ni-immobilized soil. Application of the mixture of both amendments reduced grain Ni concentration, phytate, Phytate/Fe, Phyt/Zn molar ratios, and soil DTPA-extractable Ni. In addition, the amendment mixture increased 70 % Fe and 229 % ferritin concentrations in rice grains grown in the soil at original pH. The Fe and ferritin concentrations in S-treated soil was increased up to 113 and 383 % relative to control respectively. This enhanced Fe concentration and corresponding ferritin in rice grains can be attributed to Ni/Fe antagonism where Ni has been immobilized by GS and BC mixture. This proposed technique can be used to enhance growth, yield, and Fe biofortification in rice by reducing soil pH while in parallel in situ immobilizing Ni in polluted soil.