Enzyme-Functionalized nanomaterials to use soil ORganic P Reserves for AGriculturE – FORAGE

The expected results of the project are the production of «innovative« fertilizers containing one or more enzymes depending on the type of soil to be «fertilized« and the forms of Po it contains.
The first step is to produce large quantities of enzymes that are not marketed. For this, recombinant technologies will be used. Then, for each candidate enzyme free in solution, their substrate specificities will be established, in the presence and in the absence of soil. Different inositol-phosphate molecules, varying from a phosphate group (IP1) to 5 phosphate groups (IP5) on the 6 possible positions (IP6 = phytate, reserve form of P in seeds and main form of Po in soil) will be synthesized during the project in order to better understand the capacity of candidate enzymes to hydrolyze these different substrates. Different enzyme encapsulation processes will be tested in order to find those which will be (i) the most effective to protect the enzyme, (ii) which will allow maximum enzymatic activity in solution and under soil conditions and (iii) which will allow keep the activity of the enzyme as long as possible under industrial storage conditions. Finally, new industrial processes will be developed in order to manufacture innovative fertilizers from encapsulated enzymes. The final stage of the project will be to test the effectiveness of these new enzyme-based fertilizers on the nutrition P of species of agronomic interest, grown in pots.

Three phosphatases from different microorganisms (bacteria, fungi, etc.) with contrasting specificities and affinities with respect to their different substrates were produced successfully. A fourth plant-derived phytase is in production.
Among the salient results, we have observed that two of the enzymes are very specific for phytate while the other hydrolyzes many organic P compounds except phytate. In addition, it is observed that the different enzymes hydrolyze phytate or other organic P compounds with the same efficiency in the presence of different cations, and this has never been shown. These results are very important for the hydrolysis of organic P under soil conditions, as it is most likely associated with cations like calcium.
The three enzymes have been successfully incorporated into hybrid materials (silica-phospholipids, patent WO2017006046 - PCT / FR2016 / 051701) after modifications to the manufacturing process of the materials to better accumulate and conserve the enzyme in the materials. The materials produced were then studied in more detail. Among the properties studied, the ability of these materials to hydrolyze phytate (exogenous) to Pi in unsterilized soils was quantified. The first results obtained show that there is indeed production of Pi (assayed by removing Pi thanks to anion exchange resins) in the soils.
Finally, two first models of partially phosphorylated inositols (IP2 and IP5) have just been produced.

The first results indicate that the chosen strategy - production of recombinant enzymes and encapsulation in nanomaterials - effectively makes it possible to release mineral P (Pi) from organic P in the soil which could therefore be absorbed by the roots and therefore contribute to the crop nutrition P. However, it remains to validate the production of innovative fertilizers in the form of granules from nanomaterials functionalized by the various enzymes.

Not applicable

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.