RESUMEN
Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii ("bianchetto" truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This "green" method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.