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Digital microfluidics for growing unculturable microorganisms

Identifieur interne : 000007 ( Hal/Curation ); précédent : 000006; suivant : 000008

Digital microfluidics for growing unculturable microorganisms

Auteurs : Mathieu De La Motte Saint Pierre [France]

Source :

RBID : Hal:tel-02337717

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Abstract

Soil is the natural medium containing the highest microbial diversity (109 cells from 104 different species per gram of soil). Yet we still can’t grow in laboratory more than 5 % of them. Having access to this diversity will lead to crucial applications for farming (production of organic fertilizers or environmentally friendly pesticides) and to pharmacology (discovery of new antibiotics or new anticancer molecules). This work focuses on the study of growth of non culturable micro-organisms from natural samples, like soil. This method uses microfluidics droplets as microreactors to obtain the growth of microbial species encapsulated inside. The first step is to achieve a solution with nothing but the microbes from our natural sample (no minerals) for a successful encapsulation and obtain diversity as close to the one found in the soil. The second step is to encapsulate the cells from this solution with different set of condition like : initial concentration, growth media and incubation time. By coupling observation of the droplets after growth and the rRNA 16S sequencing of their content we demonstrate that it is possible to obtain the growth of up to 40 % of the species. This microfluidic method, besides its use in growing unculturable species in laboratory, opens the way towards high-throughput screening of interactions between a given species (human or plant pathogens, phage/virus) and the microbiota it is likely to contaminate (gut flora, soil, sea …) and obtain the quantitative determination the reaction of microbiota.


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Hal:tel-02337717

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<p>Le sol constitue le milieu naturel contenant la plus grande diversité de micro-organismes (typiquement 109 cellules de 104 espèces différentes par gramme de terre). Pourtant il n’est possible d’obtenir la croissance que d’une fraction en laboratoire (moins de 5 %). Réaliser des cultures de cette diversité, inaccessible pour le moment, aurait des applications considérables en agriculture (création d’engrais ou pesticide biologique et respectueux de l’environnement) et en pharmacologie (découverte d’antibiotiques ou anticancéreux). Ce travail de thèse est principalement axé sur l’étude de la croissance de micro-organismes difficiles à cultiver issus d’échantillons naturels tels que les sols. Des gouttes de tailles micrométriques, créées par microfluidique digitale, sont utilisés comme microréacteurs afin d’obtenir la croissance en laboratoire des espèces microbiennes encapsulées à l’intérieur. Une première étape consiste à obtenir une solution ne contenant que les microbes provenant de notre échantillon naturel de sol pour pouvoir réaliser l’encapsulation sans matières minérales et obtenir une diversité la plus proche possible de celle du sol. Une deuxième étape consiste à encapsuler les cellules contenues dans notre solution en faisant varier certaines conditions comme : la concentration initiale de microbes, le milieu de culture ou le temps d’incubation. Par l’observation des gouttes après croissance et séquençage des gènes ARNr 16S des cellules contenues à l’intérieur nous démontrons qu’il est possible d’obtenir la croissance de jusqu’à 40 % des espèces. Cette méthode microfluidique ouvre la voie du criblage à haut débit des interactions entre une espèce donnée (pathogène humain ou de plante, phage/virus) avec le microbiote qu’il est susceptible de contaminer (flore intestinal, sols, mers …) et ainsi déterminer quantitativement la réaction du milieu étudié, en plus de son utilisation pour la croissance d’espèces difficilement cultivables en laboratoire.</p>
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