Abstract
Soil is an extremely heterogeneous matrix where organic and inorganic phases coexist with a variety of living organisms. In particular, the rhizosphere is a very dynamic environment where plants and microorganisms compete (or cooperate) for mineral nutrients. In such a dynamic system, thermodynamic previsions about chemical processes can be controverted by peculiar conditions, which may kinetically favor other unexpected reactions. It is widely accepted that low-molecular-weight organic acids released into the rhizosphere have complexing properties thereby enhancing the solubility of several mineral elements and increasing their availability for plant uptake. Among these, it is well known that citrate can increase mineral nutrients mobility (particularly Fe), especially in alkaline soils. However, experimental evidence showed that, in a calcareous soil (pH 8.2, CaCO3 61.8% w/w, Corg 0.86% w/w), citrate (0.1 mM and 1 mM) was not as effective in solubilizing Cu as Al, Fe and Mn from soil. In addition, Automated Particle Analyses (APA) using Scanning Electron Microscopy coupled to Energy Dispersive X-ray spectroscopy (SEM-EDX) and Cluster Analysis evidenced the formation of a large number of aluminum oxide (AlOx) particles, about 50% of which containing a significant amount of Cu. In this research we demonstrated that in particular conditions of alkalinity and high microbial activity, which can be typically found in the rhizosphere of plants grown in calcareous soils, citrate efficacy in mobilizing Cu is strongly reduced by co-precipitation of the metal within Al (hydr)oxides. To prove this hypothesis, conditions similar to those occurring in the real soil (100% calcite, pH 8.2, [Al]=17 mg/l, [Cu]=7 mg/l) have been simulated, at first, by gradually reducing the concentration of citrate and thus mimicking the action of soil microorganisms feeding on citrate (as also observed experimentally) and, in a second step, by introducing soil microorganisms into the system. As a result, in both cases we observed a significant and concomitant decrease of Al and Cu solubility; then APA analyses on the solid phase revealed the formation of Cu-containing Al (hydr)oxides almost identical (in size, shape and composition) to those observed in the real sample. In addition, 16S rDNA PCR-DGGE of DNA extracted from the samples revealed changes in microbial communities suggesting that specific bacteria may be responsible for the degradation of citrate-metal-complexes. Sequencing of the main DNA gel bands allowed identification of these bacteria as Sphingomonax sp., Sphingopyxis sp. and Propionibacterium sp. In conclusion, in highly calcareous soils, citrate can be ineffective in mobilizing Cu from soil because specific soil microbes can degrade Al-citrate complexes thus causing the co-precipitation of soluble Cu within Al-(hydr)oxides and making it unavailable for plants.