FORAGE

FORAGE aims at developing new products in fertilizer industry based on nanomaterials functionalized with enzymes to improve the supply of phosphate to agriculture from the huge soil organic P (Po) reserves that are unaccessible to plants. To be accessible, the various molecules composing the Po pool need to be hydrolysed by soil enzymes and liberate orthophosphate, the major P compound able to be uptaken by plant roots. The interest of this technological approach comes from the warning by many agronomists and economists of a shortage of mineral P (Pi) production from resource deposits around the world. The problem has worldwide implications, but is even worst for European agriculture since very few P deposits are present in Europe, making it highly dependent on imports from foreign countries and moving it away from an objective of global food self-sufficiency in a politically changing world. As Po represents a very important fraction of total P in soils (between 30 and 90%, depending on soil type and land use), the main objective of this project is to accelerate the P biogeochemical cycle by an increased efficiency of the mineralization of Po to Pi. For this purpose, we will use recombinant technologies to produce 4 enzymes targeting (i) a wide range of Po compounds via an ectomycorrhizal fungal acid phosphatase (Hebeloma cylindrosporum) or (ii) phytate, the main form of soil Po, via a fungal (Aspergillus niger), a bacterial (Bacillus subtilis) or a plant phytase (Purple Acid Phytase) to cover a range of cations/metal-phytate forms and soil pH. These enzymes are not commercially available in sufficient quantities for a full evaluation of their efficiency in agronomical assays and they will be produced in fermenters. The optimization of the production in fermenters will be studied using recombinant Pichia pastoris clones secreting these enzymes, before being carried out in an industrial pilot unit. The most important aspect of the efficiency of the selected enzymes on Po hydrolysis is the long-term persistence of their catalytical activity in soil. Our approach of this key aspect of the project is to study the protective effect of adsorption/encapsulation of the enzymes by mineral or organo-mineral nanomaterials. We will use NMR and chromatographic methodological approaches, along the use of several phytase synthetic substrates specifically designed for this study (partially phosphorylated inositols or fluorescent phytate) to assess the actual activity of these enzymes in soil, whether free or associated with nanomaterials. The optimization of the catalytic activity of these enzyme-functionalized nanomaterials will be achieved (i) by studies on the effect of the mineral surfaces on the potential modification of conformation of the enzymes which could decrease their efficiency at short and longer term, (ii) by taking into account the molecular diffusion of both the substrate and the product of the enzyme catalysis in an adsorbed or encapsulated state. The industrial feasibility and the effect on plant P nutrition of these new fertilizers will be established in a large scale experiment in a phenotyping platform on wheat, pea and ray-grass. The plant growth, P content, grain yield and gene expression known to be over-expressed under P stavation conditions will be studied. We will also follow an Intellectual Property process to secure these new enzyme-based fertlizers. The rentability of such technology will also be evaluated but first estimates of the cost of enzyme at an industrial scale appears to be reasonable. We expect that we could propose these new enzyme-functionalized nanomaterials as a solution to cope with the predicted shortage of mineral P resources endangering European agriculture.