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Conception d'une méthode de diagnostic fonctionnel des microbiotes du sol en agriculture grâce à la millifluidique de gouttes

Abstract : Soil microbes are useful in agriculture: they help plants to acquire nutrients, degrade waste, protect crops against pathogens, etc. Understanding the interactions between microorganisms, plants and other soil elements could help increase productivity while preserving the environment. From this perspective, one of the challenges in agronomy is to be able to characterize soil microbial communities in a functional manner; that is, to quantify the services they can render to agriculture and society in general. For example, a "good" microbiota from a functional point of view will protect plants from diseases and help them acquire nutrients, degrade harmful pollutants and limit the influx of greenhouse gases. To carry out this characterization, the identification and genetic analysis of microbiotes is not sufficient; technological tools are lacking.We chose to address this problem by exploiting a technology that has not been used in agriculture until now: droplets millifluidics, which consists in manipulating drops of a few hundred nanoliters, separated from each other by an oily phase. In this case, we used the automated microbiological drop culture system from MilliDrop, a startup from our lab. We studied one function: the ability to solubilize soil phosphate, an essential plant nutrient. We adapted the recipe of a culture medium used for more than 70 years: Pikovskaya medium. It contains particles of calcium phosphate and we had to carry out an important physicochemical formulation work to transfer these particles into drops and keep them dispersed. We also added two fluorescent probes to the medium that allowed us to monitor both the pH of our drops and the respiratory activity. Once the experimental protocol was ready, we applied it to a dozen agricultural soils.Thanks to the fluorescence of resazurin, our respiratory activity probe, we estimated the concentration of cultivable cells in our samples. We showed that the same number of microorganisms was obtained in trend with our drop method as with the classical method of counting colonies on Petri dishes.By using an optical block designed for our project, we have been able to conduct optical scattering measurements in drops and evaluate the ability of our microorganisms to lower this signal, which we interpret at least partially as the ability of the microorganisms to solubilize phosphate. Thanks to our pH probe, we were able to show that the fall of the scattering signal was associated with a pH fall below 5.8 (which corresponds to the theoretical pH below which particles dissolve by the sole effect of acidity) in about 90% of the drops. It is possible that among the remaining 10% of the drops there are microorganisms that secrete large quantities of chelating agents, which is of particular agricultural interest. There are systematically more solubilizing microorganisms in the drops than with the traditional Petri dish method, but there is no correlation between these two methods of measurement. While different hypotheses could be tested to clarify this phenomenon, our results call into question the traditional test on plates.Our protocol is quick and simple: our experiments were carried out by a non-specialized technician; data processing can be automated, and half an hour of work is enough to analyze four soils (more with future versions of the machine). The results in drops are obtained seven times faster than those on Petri dishes. This makes our protocol a candidate to become a functional test used on a large scale.
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Submitted on : Thursday, May 6, 2021 - 2:36:13 PM
Last modification on : Thursday, April 7, 2022 - 10:10:44 AM
Long-term archiving on: : Saturday, August 7, 2021 - 7:06:19 PM


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  • HAL Id : tel-03219526, version 1



Arthur Goldstein. Conception d'une méthode de diagnostic fonctionnel des microbiotes du sol en agriculture grâce à la millifluidique de gouttes. Chimie analytique. Université Paris sciences et lettres, 2021. Français. ⟨NNT : 2021UPSLS038⟩. ⟨tel-03219526⟩



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