Periodic Reporting for period 3 - SENSOILS (Sensing soil processes for improved crop nitrogen bioavailability)
Reporting period: 2018-09-01 to 2019-12-31
The recent development of transparent soils in my group gives great scope to unravel the processes involved in the reactive transport of nutrients in soil and their interaction with the soil biota. The broad aim of this project is to exploit and further develop the technology and understand the role of chemical and microbial process involved in nutrient movement at the micro-scale. We expect this research will not only shed new lights on what limit the efficient transfer of nutrients to crops, but also deliver fertiliser screens that more informative, improve the testing of new generation of fertilisers and promote the development of future fertilizers with less impact on the environment. New model soil systems could be used to better understand the spread of soil borne diseases, the bio-remediation of contaminated soils and the mechanisms underlying soil biodiversity and activity.
(WP1) – New generation of smart soil particles
The objective of this work package is to develop new types of artificial transparent soils for both the measurement of biological activity and the measurement of nutrient concentration in the rhizosphere. In order to achieve this objective, we have adopted a core-shell strategy for the design of soil particles. The core of the particle is composed of a material chosen for its low refractive index and transparency. The core material must also be amendable to attachment and coating by the active polymer so that a thin shell is built around the core particle. In order to achieve this objective, and as fluoropolymers are well known for their transparency, we chose to use a copolymer based on tetrafluoroethylene and hexafluoropropylene (called Fluorinated Ethylene Propylene, FEP) instead of Nafion® as core material for the particles. Actually, FEP is not as transparent as Nafion®, but it is of lower cost, can be easily sourced from commercial suppliers, has a refractive index closer to water and its chemistry is more amendable to interactions with other fluorinated polymers. The shell must also be made of a low refractive index polymer, transparent, but it must incorporate numerous functions such as ion exchange, chemical sensing, and possible adhesion.
A number of key milestones have been reached toward the making of sensing soil particles. These include porotocols for the making of FEP cores. FEP is quite different from Nafion because the temperature at which it becomes more ductile is lower than Nafion. Obtaining suitable particle size with FEP particles is therefore much harder than with Nafion. We have successfully attach various polymer and co-polymer on the surface of FEP membranes and particles. FEP has been developed originally as a non-sticky inert material and to date this is the first time such coatings have been achieved. The coating was made of novel terpolymers based on 1,1,1,3,3,3-hexafluoropropyl a-fluoro acrylate (FAHFiP), 2-(trifluoromethyl) acrylic acid (MAF) and fluorescein 2-(trifluoromethyl) acrylate (MAF-fluorrescein).
The ability to attach fluorinated terpolymers on FEP core particles was also followed by several breakthroughs in the synthesis of such fluorinated copolymers which structure and composition are being tailored to obtain suitable physical and chemical properties. Our terpolymers are based on either 2,2,2-trifluoroethyl α-fluoroacrylate (FATRIFE) or 1,1,1,3,3,3-hexafluoroisopropyl α-fluoroacrylate (FAHFiP) backbones to which various other monomers are introduced for control of hydrophilic behaviour, ion exchange, fluorescent marking, sensing, etc. Original block copolymers that display a sequence adhesive to FEP and the other one hydrophilic have been obtained via RAFT sequenced copolymerization. Control of hydrophilic behaviour and ion exchange of the co-polymers are achieved through incorporation of 2-(triuoromethyl)acrylic acid (MAF) groups in the terpolymers. To evidence such a trend, we first focused on the synthesis of poly(MAF-co-FATRIFE) copolymers both of them inducing complementary properties. These copolymers showed that increasing the MAF concentration in copolymers also induced a reduction of the contact angle of polymer coatings and thus a hydrophilicity (a first article was published in Polym. Chem. Banerjee et al. 2017). We have also successfully included Fluorescein in our co-polymers and this demonstrates the possibility of obtaining constitutive fluorescence. The fluorescein coatings were successfully detected by various microscopes and plant growth was successfully tested on these new particles, although water retention (hydrophilicity is still not sufficient). Importantly, work has also started to introduce sensors in the shell co-polymers. We have decided to investigate pH sensing molecules because of their abundance in the literature. As part of this effort, we have successfully introduced Nile blue, a pH sensing dye which responses to pH cover a broad spectrum (Scheme 3).
(WP2) – Microscopic processes at the surface of soil particles
The objective of the second work package (WP2) is to develop a series of experiments to understand key processes at the surface of the soil particle. The first stage of WP2 has focused on the development of a biological model which complement the work of WP1 focusing on the chemistry of soil particles.
Work has started to develop a model system for nitrification in the rhizosphere. In particular, a strain of Nitrobacter has been obtained (Nitrobacter hamburgensis X14, DSMZ). Culture of the bacteria was started (Growth in 756a medium in mixotrophic conditions at 28°C). Transformation has also been tested although it has not been successful so far. Methods tested include Electroporation and Nanoparticles transformation.
Because pH dyes are common and because we have identified more easily candidates for co-polymerisation (work package 1), we also focused on studying the microbial response mechanism for adaptation to switch in pH in the rhizosphere. This allows WP2 to align with the development of pH sensing soils, but also to work on microbes with genetic transformation and widely available genetic resources. Preliminary experiments have started with Pseudomonas fluorescens and Bacillus subtilis. Work has characterised bacterial adherence during the early stages of root development and the effect of the presence of root exudate bacterial growth and movement. Transformation of Pseudomonas fluorescens with fluorescently labelled plasmids (red and green) and acquisition of GFP Bacilus subtilis strains has prepared the model system for live imaging. The challenge now is to determine which plant and bacterial factors controls establishment in the rhizosphere community and relating quantitative data from microbiological assays with that seen in live images. The next stage is to incorporate a reporter for intra cellular pH variation and to study how changes in external pH affect the growth of these bacterial organisms.
(WP3) – Rhizosphere formation
The objective of this work package is to understand how, where and when root exudates modify microbial activity during the establishment of the rhizosphere, and how this contributes to nutrient movement in soil. Process in the rhizosphere operates at a larger scale (mm to cm scale) and over longer time period. Work has started to establish protocols and tools to carry out quantitative imaging data at this spatial and temporal scale and planning for experiments and hypothesis testing has started.
We have achieved key progress in our ability to carry out time lapse imaging in transparent soil. These advances are based on the development of a third generation of growth chambers based on microfluidics technologies. The chambers use laser cut casts for shaping Polydimethylsiloxane (PDMS) parts holding microscope coverslips; 3D printed tools for precision assembly of the different parts of the chambers, and oxygen plasma for assembly, sterilisation and surface treatment for hydrophilic behaviour of the chamber. Secondly, we have made good progress for the control of water content in soil with the design of porous PDMS parts where soil water tension can be controlled . A live imaging dedicated to imaging the rhizosphere has been developed. The system uses dual illumination light sheet system based on Powell lens and multi wavelength illumination system (50 mW, 488 - 514 - 561 - 633 nm collinear). Custom made LED illumination system, movement and software for the control of the different parameters of the experiments have been constructed.
Preliminary experiments have been carried out using Lettuce (Lactuca sativa) and Pseudomonas fluorescens as a model system, assays have been developed measuring factors of early colonisation such as bacterial motility, chemotaxis and adherence to roots. Data from these assays will be supplemented with live imaging of fluorescently marked bacteria in transparent soil, providing information on bacterial localisation on roots. A main challenges now it the integration of WP1&2. There has been excellent progress with the development of techniques to allow whole rhizosphere imaging and to obtain quantitative image data. This work package now relies essentially on progresses from WP 1 & 2 to fully deliver the key objectives set by the grant. The main remaining challenges will then become the integration of the various compounds from the previous work packages 1&2 into complex “system” experiments.
(WP4) – Modelling the rhizosphere
The aim of this work package is to construct a theoretical framework to model the rhizosphere, from the time a root tip enters the bulk soil until maturity of the root tissue. At the microscopic scale, the rhizosphere can be viewed as an ensemble of interacting microscopic elements, namely, bacterial cells, soil particles, root cells. Hence, it was decided to base the theoretical framework on the Smooth Particle Hydrodynamics (SPH) method. SPH allows irregular arrangement of particles (cells, particles) to be modelled and also to integrate physical behaviour in a very efficient way. Initial work focused on applying SPH techniques to model root growth.
We have developed a system (mathematical model and simulations programs) that allows multiple and complex interactions taking place between root and multiple types of microbes. Interactions include variable exudation rates, microbial growth, mobility, attachment, root elongation rate and profile. Because models are solved in a one dimensional moving reference frame, the framework is computationally very efficient and is amendable to various forms of additions and complexification. The model was published recently (Dupuy & Silk 2016), and is being used for fast testing of hypothesis.
A key achievement of this work package however, is that we have developed the first SPH formulation of the growth and mechanics of plant tissue. The model represents the tissue as a poro-elastic material, with the solid phase being the cell wall architecture and the pore pressure representing turgor pressure. The new model was implemented in a community driven open-source code, DualSPHysics (http://dual.sphysics.org/). In the coming months, we expect to be able to run simulation of entire root tips at cellular resolution. This has never been achieved to date.
First, the development of core shell structure technologies to make sensing soil particles or ""Sensing soils”. These will have a broad range of application, for example in scientific research, breeding and development of fertiliser. It has required significant advancement in polymer sciences, and the techniques could be used in other areas of science and technology.
Our work is greatly contributing to the field of soil microscopy. We have developed a series of approaches that allow live observation of soil microbes. We fabricated new fluidics systems for soil control of soil conditions under a microscope.
Computational modelling work for simulation of root development in soil is extremely promising. We anticipate we will be possible to make simulation of entire roots at cellular resolutions while at the same time model the soil at the particle level. The model and algorithms are being implemented in DualSPHysics, an open source community led simulation tool.
Work focused on understanding factors affecting microbial activity in the rhizosphere is, and is just starting to benefit from the technological development from the project. In particular, we expect to be able to quantify accurately the mobility coefficient of soil microbes surrounding plant roots and predict what contributes to maintenance of certain microbes at the tip of the root.