Designing of novel hydroxyapatite nanoparticles from fish by-products to be coupled with highly efficient phosphate solubilising bacteria

Hydroxyapatite nanoparticles (nHAs) deriving from by-products have gained increasing interest as novel phosphorus (P)-based fertilisers, since they can provide a slow P release, minimising P losses and adverse environmental side-effects, and reducing the dependency of agriculture on mineral fertiliser inputs. Phosphate solubilising bacteria (PSB) have proven to release P available for crop uptake from different inorganic sources (e.g. tricalcium phosphate, TCP, hydroxyapatite, HA). In the present study, nHAs were prepared from salmon (S-nHAs) and tuna (T-nHAs) bones by a calcination process, followed by a high energy ball milling. The obtained fine powders were characterised by scanning electron microscopy (SEM) for size and shape and by X-ray diffraction (XRD) for crystal phase composition. The phosphate solubilisation activity of seven selected PSB strains belonging to Pseudomonas and Paraburkholderia genera was in vitro investigated under acidic (pH = 5.5) and alkaline (pH = 7.5) conditions by a quantitative assessment of the solubilised PO₄^3- from TCP, S-nHAs and T-nHAs over time. Moreover, time trend of pH and organic acids in the liquid media were investigated. Characterization of S-nHAs by XRD and SEM revealed a biphasic composition of the material consisting of TCP and HA – about 50 wt% of each phase - and a heterogeneous rounded-shape (Ø < 50 nm) material. By contrast, XRD pattern of T-nHAs showed a single-phase composition mainly made of pure HA (> 95 wt%) and SEM micrographs exhibiting an elongated shape uniform in size (200 x 30 nm). At day seven, Pseudomonas graminis PG0319 solubilised the highest proportion of the total PO₄^3- in the TCP substrate under acidic pH (83%), followed by Pseudomonas rhodesiae PR0393 and P. graminis PG1211 (79% and 72%, respectively). In S-nHAs under alkaline pH, Paraburkholderia terricola PT0405, PR0393, PG0319 and PG1211 solubilised from 53% to 57% of the total PO₄^3-, whereas in T-nHAs under acidic pH the maximum solubilisation efficiency was 27% by PT0405 at day seven. The difference in the solubilisation of S-nHAs and T-nHAs is due to the lower solubility of HA in comparison with TCP. Values of pH in in the liquid media decreased over the time along with an increasing PO₄^3- solubilisation activity, suggesting an extracellular secretion of organic acids by PSB. Accordingly, differential patterns of organic acids were detected among strains with TCP as well as S-nHAs and T-nHAs. Notably, gluconic, propionic, fumaric and acetic acids played key roles during P solubilisation with all the tested strains, substrates, and pH conditions. Our results indicate that the use of microbial inocula together with P-based nanofertilisers is a promising option for a sustainable agricultural transition.