WO2016034714A1

WO2016034714A1 - Capsules with a conductive membrane

Info

Publication number: WO2016034714A1
Application number: PCT/EP2015/070266
Authority: WO
Inventors: Léopold MOTTET; Jérôme Bibette; Nicolas Bremond; Philippe Poulin
Original assignee: Espci Innov
Priority date: 2014-09-04
Filing date: 2015-09-04
Publication date: 2016-03-10
Also published as: FR3025437B1; FR3025437A1

Abstract

The invention relates to a capsule consisting of a liquid core and at least a gelled envelope entirely encapsulating the liquid core on the periphery thereof, said gelled envelope comprising an electroconductive material. The invention also relates to the use of such a capsule for producing bioreactors, screening micro-organisms or producing an anode and/or cathode of microbial fuel cells, to a method for screening electroactive bacteria using said capsule, and to a device comprising such a capsule.

Description

Conductive membrane capsules

The present invention relates to a capsule formed of a liquid core and at least one gelled envelope completely encapsulating the liquid core at its periphery, said gelled envelope comprising an electrically conductive material; the use of such a capsule for the production of bioreactors, the screening of microorganisms or the production of an anode and / or cathode of a bacterial cell and a method for screening electroactive bacteria using said capsule and a device comprising such a capsule.

The limitation of the production of waste and the question of their valorization, and more particularly that of the valorization of their energy, is today a major concern.

At present, the European Union is investing heavily in waste treatment.

In addition, wastewater treatment in the United States generates 15 GW of energy ($ 25 billion), which represents 3% of US electrical power.

Household, industrial or animal waste thus represents a potential energy output of 17 GW and an energy of 5.10 ¹¹ kWh.

Microbial cells, also known as bacteria cells, are remarkable green energy devices that exploit microorganisms to produce electricity from organic compounds such as waste. The bacterium feeds on waste and at the same time releases electrons.

However, current bacterial cells have certain drawbacks, and there is a need for improvement of energy production by existing bacterial cells, particularly by the search for more electrochemically active microorganisms.

The selection of electroactive organisms is currently done by parallelizing cultures in micro-wells with an electrode (Hou, Han, & al PlosOne August 2009). In a near field, high-throughput enzyme selection is done through 96-well plates (Song, O'Donohue Bioresource Technology 2010).

The inventors of the present invention have developed a capsule that can encapsulate an electro-active object that has many advantages over the prior art techniques for selecting electroactive microorganisms. The capsule according to the invention makes it possible to compartmentalize the culture and therefore to decorrelate the bioreactor from the reading system of the electrochemical activity of the microorganisms. It also allows a culture over a longer time because the membrane of the capsule protects the culture from contaminations. In addition, and contrary to what is observed with micro-well cultures, the culture in capsule allows the renewal of the medium without complex manipulations such as centrifugations or multiple pipetting. The easy renewal of the environment eliminates the problems related to evaporation.

Moreover, the capsule according to the invention makes it possible to connect the culture to a reading system to know the electrochemical performances of the encapsulated organism.

Thus, the capsule according to the invention provides a new tool to the culture of microorganisms in liquid heart capsules by allowing to act electrically on the encapsulated organism.

The present invention therefore relates to a capsule comprising a liquid core, a gelled envelope completely encapsulating the liquid core at its periphery, the gelled envelope being adapted to retain the liquid heart when the capsule is immersed in a gas, an aqueous solution or an oil, the gelled envelope comprising at least one gelled polyelectrolyte and at least one surfactant, characterized in that the gelled envelope further comprises an electrically conductive material.

By "electrically conductive material" is meant a material capable of passing an electric current, that is to say electrons, and convey the current, so electrons, from one point to another, through a solid medium. It is understood in the context of the present invention that the electrical conductivity covers all types of electrical conductivities, with the exception of the ionic conductivity, defined by the displacement of ions in a liquid. Thus, in the context of the present invention, the electrically conductive material is not an ionically conductive material.

In particular, the electrically conductive material consists of electrically conductive solid particles, more particularly of carbon nanotubes, carbon black or graphene.

Even more particularly, said electrically conductive material is biocompatible.

By "biocompatible material" is meant a material that does not degrade the biological medium in which it is used. A biocompatible compound according to the invention is for example a polyelectrolyte of natural origin such as chitosan or sodium alginate.

The liquid heart of the capsule according to the invention consists of an aqueous or oily liquid that may contain molecules, microorganisms, or other objects larger than the maximum pore size of the membrane (about 20 nm).

According to one aspect of the present invention, the liquid core comprises at least one electro-active object.

By "electro-active object" is meant an electrically active or reactive object capable of exchanging, generating or capturing electrons or generating or sensing another electro-active object.

In particular, the electro-active object is chosen from microorganisms, cells, macromolecules and electroactive solid particles. Macromolecules are molecules with at least several tens of atoms. By "macromolecule" is meant for example a protein, a lipid, a sugar or a polymer. The term "electroactive solid particles" means particles onto which electroactive objects such as microorganisms, cells or macromolecules defined within the scope of the present invention are grafted or adsorbed. The particles on which electroactive objects are grafted or adsorbed are, for example, particles of gold, polystyrene or silica.

Even more particularly, the electro-active object is selected from bacteria of the genus Escherichia Coli, Geobacter, Shewanella, Rhodopseudomonas, Ochrobactrum, Enterobacter.

In the context of the present invention, the liquid heart of the capsule may comprise at the time of encapsulation variable amounts of molecules, microorganisms, macromolecules and cells that will be adapted depending on the type of encapsulated object.

For example, the liquid heart of the capsule may comprise at the time of encapsulation an amount of cells greater than or equal to 1 cell / capsule.

It may also comprise at the time of encapsulation an amount of microorganisms greater than or equal to 1 microorganisms / capsule, preferably greater than the minimum inoculum. For each bacterium, there is indeed a minimal inoculum that corresponds to the minimum concentration of bacteria that allows the crop to grow.

Preferably, the liquid contained in the liquid heart is physiologically acceptable. It may in particular comprise a saline solution, macromolecules or molecules, a physiologically acceptable viscosifier and / or a culture medium for the growth and / or culture of microorganisms or cells.

By "culture medium" is meant any medium, in particular any liquid medium, capable of supporting the growth of cells or microorganisms. For the encapsulation of bacteria, use is made, for example, of a core composed of 15% of PEG3500 and 0.45% of NaCl. this saline solution for maintaining an acceptable osmolarity for the microorganism during encapsulation, this osmolarity being similar to that of the culture medium of the bacterium.

The liquid heart may also include physiologically acceptable excipients, such as thickeners, or rheology modifiers. These thickeners are, for example, polymers, cross-polymers, microgels, gums or proteins, including polysaccharides, celluloses, polysaccharides, silicone-based polymers and co-polymers, colloidal particles (silica, clays). , latex, biocompatible carbon nanotubes ...).

The gelled envelope of the capsules according to the invention comprises a gel containing water, at least one polyelectrolyte reactive with multivalent ions and the electrically conductive material. According to the invention, the envelope further contains a surfactant resulting from its manufacturing process, as described below.

In particular, the envelope contains at least one surfactant, preferably two.

Thus the present invention also relates to a capsule as mentioned above, comprising two surfactants.

In particular, the first of the two surfactants allows the formation of the capsule. It may for example be chosen from sodium dodecyl sulfate or sodium stearoyl-2-lactylate.

In particular, the second of the two surfactants makes it possible to stabilize the dispersion of the electrically conductive material. It may for example be chosen from sodium dodecyl sulfate, polyoxyethylene (23) lauryl ether or polyoxyethylene (20) cetyl ether. More particularly, its dialysis then allows the destabilization of the dispersion and the creation of a conductive network.

In particular, the capsule according to the invention is obtained from a process comprising the following steps of:

a) separate conveying in a double jacket of a first aqueous or oily liquid solution and a second liquid solution containing a liquid polyelectrolyte suitable for gelling and the electrically conductive material; b) forming, at the exit of the jacket, a series of drops, each drop comprising a central core formed of said first solution and a peripheral film formed of said second solution and completely covering the central core;

c) immersing each drop in a gelling solution containing a reagent capable of reacting with the polyelectrolyte of the film to change it from a liquid state to a gelled state and form the gelled envelope comprising the electrically conductive material, the central core forming the liquid heart;

d) recovery of formed capsules;

the second solution containing at least one surfactant prior to contact with the first solution and therefore before contact with the gelling solution.

More particularly, said second liquid solution containing a liquid polyelectrolyte suitable for gelling and the conductive material mentioned in step a) of the process is formed by contacting the electrically conductive material in dispersed form and the polyelectrolyte suitable for gelling.

Even more particularly, the gelled envelope is formed by a divalent ion gelled polysaccharide hydrogel comprising electrically conductive solid particles in dispersed and connected form thereby forming an electrically conductive network.

According to one aspect of this method, the ratio of the flow rate of the first solution to the flow rate of the second solution at the outlet of the double jacket is between 0.1 and 20, advantageously between 1 and 5, the gelled envelope having a thickness of between 1 .6% and 55%, advantageously between 6% and 20% of the diameter of the capsule, after recovery of the capsules formed.

In another aspect of the process, the drops formed by coextrusion in the jacket fall by gravity through a volume of air into the gelling solution.

In the context of the present description, the term "surfactant" means an amphiphilic molecule having two parts of different polarity, one lipophilic and apolar, the other hydrophilic and polar. A surfactant may be of ionic type (cationic or anionic), zwitterionic or nonionic.

The surfactant is preferably an anionic surfactant, a nonionic surfactant, a cationic surfactant, or a mixture thereof. The molecular weight of the surfactant is between 150 g / mol and 10,000 g / mol, advantageously between 250 g / mol and 1500 g / mol. In the case where the surfactant is an anionic surfactant, it is for example chosen from an alkyl sulphate, an alkyl sulphonate, an alkyl aryl sulphonate, an alkaline alkyl phosphate, a dialkyl sulphosuccinate, an alkaline earth salt of saturated or unsaturated fatty acids. These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbons greater than 5 or even 10 and at least one hydrophilic anionic group, such as a sulphate, a sulphonate or a carboxylate linked to one end of the hydrophobic chain.

In the case where the surfactant is a cationic surfactant, it is for example chosen from an alkylpyridium or alkylammonium halide salt such as n-ethyldodecylammonium chloride or bromide, cetylammonium chloride or bromide (CTAB) . These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbons greater than 5 or even 10 and at least one hydrophilic cationic group, such as a quaternary ammonium cation.

In the case where the surfactant is a nonionic surfactant, it is for example chosen from polyoxyethylenated and / or polyoxypropylenated derivatives of fatty alcohols, fatty acids, or alkylphenols, arylphenols, or from alkyl glucosides, polysorbates, cocamides.

In particular, the surfactant will be chosen from the following list: an alkyl sulphate, an alkyl sulphonate, an alkyl aryl sulphonate, an alkaline alkyl phosphate, a dialkyl sulphosuccinate, an alkaline earth salt of saturated or unsaturated fatty acids, a salt of alkylpyridium or alkylammonium halides such as n-ethyldodecylammonium chloride or bromide, cetylammonium chloride or bromide, polyoxyethylenated and / or polyoxypropylenated derivatives of fatty alcohols, fatty acids or alkylphenols, or from arylphenols, alkyl glucosides, polysorbates, cocamides or mixtures thereof.

More particularly, the total weight percent of surfactant in the second solution will be greater than 0.01% and is advantageously between 0.01% and 0.5% by weight.

For the purposes of the present invention, the term "polyelectrolyte reactive with polyvalent ions" means a polyelectrolyte capable of passing from a liquid state in an aqueous solution to a gelled state under the effect of contact with a gelling solution containing multivalent ions such as ions of an alkaline earth metal chosen for example from calcium ions, barium ions, magnesium ions.

In the liquid state, the individual polyelectrolyte chains are substantially free to flow relative to one another. An aqueous solution of 2% by weight of polyelectrolyte then has a purely viscous behavior with shear gradients characteristic of the shaping process. The viscosity of this zero shear solution is between 50 mPa.s and 10,000 mPa.s, advantageously between 1000 mPa.s and 7000 mPa.s.

The individual polyelectrolyte chains in the liquid state advantageously have a molar mass greater than 65000 g / mol.

In the gelled state, the individual polyelectrolyte chains form, with the multivalent ions, a cohesive three-dimensional network that retains the liquid core and prevents its flow. The individual chains are held together and can not flow freely relative to each other. In this state, the viscosity of the formed gel is infinite. In addition, the gel has a threshold of stress to the flow. This stress threshold is greater than 0.05 Pa. The gel also has a modulus of elasticity that is non-zero and greater than 35 kPa.

The three-dimensional gel of polyelectrolyte contained in the envelope traps water and the surfactant.

The polyelectrolyte is preferably a biocompatible polymer that is harmless to the human body. It is for example produced biologically.

Advantageously, it is chosen from polysaccharides, synthetic polyelectrolytes based on acrylates (sodium, lithium, potassium or ammonium polyacrylate, or polyacrylamide), synthetic polyelectrolytes based on sulfonates (poly (styrene sulfonate)). sodium, for example). More particularly, the polyelectrolyte is selected from an alkaline earth alginate, such as sodium alginate or potassium alginate, gellan or pectin.

Alginates are produced from brown algae called "laminar", referred to as "sea weed".

Such alginates advantageously have a content of α-L-guluronate greater than about 50%, preferably greater than 55%, or even greater than 60%.

In particular, the or each polyelectrolyte will be a polyelectrolyte reactive with multivalent ions, in particular a polysaccharide reactive with multivalent ions such as an alkali alginate, a gelane or a pectin, preferably an alkaline alginate advantageously having a bulk content α-L-guluronate greater than 50%, especially greater than 55%.

More particularly, the mass content of polyelectrolyte in the second solution may be less than 5% by weight and is advantageously between 0.5 and 3% by weight.

According to one aspect of the present invention, the capsule may further comprise an intermediate envelope encapsulating completely at its periphery the liquid heart, said intermediate envelope itself being completely encapsulated at its periphery by the gelled envelope.

This liquid intermediate envelope will be formed of an intermediate composition comprising a buffer or a culture medium, and / or a viscosifier. In particular, the viscosifier will be a water-soluble polymer, such as polyethylene glycol (PEG), dextran, alginate in solution more diluted than in the outer casing or even more diluted alginate than in the outer casing mixed with an electrically conductive material.

The intermediate envelope is in contact with the heart and the outer envelope and keeps the heart out of contact with the outer envelope.

The intermediate phase is useful for stabilizing the capsule during its formation, for example in the case where the liquid core contains calcium that can induce gelation of the external phase too early. Indeed, depending on their composition, the culture media can interfere with the polymerization. It thus makes it possible to separate the liquid heart from the external phase to be gelated. It also protects the liquid heart during the formation of drops, the alginate of the outer layer which is not yet polymerized.

In particular, the liquid core and the intermediate phase being both liquid, they are eventually mixed to form the liquid heart of the capsule.

The presence of such an intermediate envelope is described in particular in the scientific article "Formation of liquid-core capsules having a thin hydrogel membrane: liquid pearls", Bremond et al, Soft Matter, 2010, 2484-2488.

Drop production

The production of the drops according to the method according to the invention mentioned above is carried out by separate conveying in a double jacket of a first liquid solution containing the aqueous or oily solution in which is preferably included one or more microorganisms, cells, macromolecules or molecules and a second liquid solution containing a liquid polyelectrolyte suitable for gelling, the electrically conductive material and at least one surfactant, the conveying being as described in WO2010 / 063937.

In the case of the additional presence of an intermediate envelope, the separated conveying takes place in a triple envelope, with a third solution comprising the intermediate solution.

At the exit of the double (or triple) envelope, the different flows come into contact and then form a multi-component drop, according to a hydrodynamic mode called "dripping" (drop-by-drop, described in particular in WO 2010 / 063937) or a mode called "jetting" (with jet instability, described in particular in FR 2012/2964017), depending on the size of the capsules desired.

The first flow constitutes the liquid heart and the second flow constitutes the liquid outer envelope. In the case of the presence of the intermediate envelope, the second flux constitutes the liquid intermediate envelope and the third flux the external liquid envelope.

According to the production method, each multi-component drop is detached from the double (or triple) envelope and falls into a volume of air, before being immersed in a gelling solution containing a reagent capable of gelling the polyelectrolyte of the outer liquid envelope, to form the outer gelled envelope of the capsules according to the invention.

According to some embodiments, the multi-component drops may comprise additional layers between the outer shell and the liquid core, other than the intermediate shell. This type of drop may be prepared by separate conveying of multiple compositions into multi-envelope devices.

Stage of qélification

When the multi-component drop comes into contact with the gelling solution, the reagent capable of gelling the polyelectrolyte present in the gelling solution then forms bonds between the various polyelectrolyte chains present in the liquid outer envelope, then passing to the state gelled, thus causing gelation of the liquid outer shell.

Without wishing to be bound to a particular theory, during the transition to the gelled state of the polyelectrolyte, the individual polyelectrolyte chains present in the liquid outer envelope are connected to each other to form a crosslinked network, also called hydrogel.

In the context of the present description, the polyelectrolyte present in the gelled outer shell is in the gelled state and is also called gelled polyelectrolyte or gelled polyelectrolyte.

A gelled outer envelope, adapted to retain the assembly constituted by the heart or the heart and the intermediate envelope, is thus formed. This gelled outer shell has a clean mechanical strength, that is to say that it is able to retain the liquid core and, in case of presence of an intermediate envelope, to completely surround the intermediate envelope. This has the effect of maintaining the internal structure of the liquid core and, if appropriate, the intermediate envelope.

The capsules according to the invention remain in the gelling solution until the outer shell is completely gelled, preferably without exceeding 60 minutes, even more preferably without exceeding 10 minutes. The gelling solution can then optionally be removed and the gelled capsules can then optionally be collected and immersed in an aqueous rinsing solution, generally consisting essentially of water, in particular of physiological saline, in order to rinse off the gelled capsules formed. This rinsing step makes it possible to extract from the gelled external envelope any excess of the reagent suitable for gelling the gelling solution, and all or part of the surfactant (or other species) initially contained in the second liquid solution.

The presence of a surfactant in the second liquid solution makes it possible to improve the formation and gelation of the multi-component drops according to the process as described above.

Characteristics of gelled capsules

Advantageously, the capsule is spherical in shape and has an outside diameter of less than 5 mm and in particular between 0.3 mm and 4 mm.

Preferably, the gelled outer shell of the capsules according to the invention has a thickness ranging from 32 μηι to 1000 μηι, preferably from 120 μηι to 400 μηι, and advantageously from 100 μηι to 350 μηι.

The capsules according to the invention generally have a volume ratio between the core and all the intermediate and outer shells greater than 1, and preferably less than 5.

According to a particular embodiment, the capsules according to the invention generally have a volume ratio between the core and all of the intermediate and outer shells of between 1 and 5.

Thus, the capsules according to the present invention are useful in many sectors of activity such as for example bioenergy, bacterial, enzymatic cells or in the manufacture of electrodes.

They find many applications such as the selection of electroactive microorganisms or the creation of electrical energy from waste.

Thus, a capsule according to the invention allows:

- high-speed production of bio-reactors (1 bio-reactor / second);

screening for microorganisms by allowing the encapsulation of a single cell which will give a mono-clonal colony, the reservoirs and therefore the cells of interest that can then be selected according to the electro-activity;

- digitalization, parallelization and miniaturization of culture;

the recovery of electrons and therefore of the energy produced by a population of microorganisms; encapsulation of electrochemical species and triggering of internal reactions in the capsule via the contact of an electrode with the membrane.

the polarization of the reactor to allow the growth of microorganisms under a given and adjustable potential

According to one of these aspects, the present invention therefore also relates to the use of at least one capsule according to the invention, for the production of bioreactors.

According to one of these aspects, the present invention therefore also relates to a method for producing bioreactors, comprising the implementation of at least one capsule according to the invention.

It also relates to the use of at least one capsule according to the invention, for the screening of microorganisms, in particular electroactive microorganisms, more particularly those selected from: Escherichia Coli, Geobacter, Rhodopseudomonas, Ochrobactrum and Enterobacter.

The capsule according to the invention can therefore be a tool for selecting electroactive organisms via the encapsulation of a single cell.

Moreover, the conductivity of its membrane allows the passage of an electrode from the inside to the outside of the capsule and vice versa. The membrane thus allows the recovery of electrons released into the external environment by the organisms in culture.

Thus, the present invention also relates to the use of at least one capsule according to the invention, for the recovery of electrons produced by microorganisms, in particular those chosen from: Escherichia Coli, Geobacter, Rhodopseudomonas, Ochrobactrum and Enterobacter.

The present invention also relates to a method for recovering electrons produced by microorganisms, in particular those chosen from: Escherichia Coli, Geobacter, Rhodopseudomonas, Ochrobactrum and Enterobacter, comprising the use of at least one capsule according to the invention. invention.

The present invention also relates to the use of a capsule according to the invention for the production of an anode and / or a bacterial cell cathode.

In particular, in the context of this use, the capsule is electrically connected to another tank.

More particularly, the two components (capsule and reservoir) are electrically connected and separated by an ion exchange membrane.

By "reservoir" is meant a volume containing a redox species and an electrode. This reservoir completes the capsule, playing the role of anode or cathode so that the assembly constitutes a bacterial cell that is to say an anode and cathode electrically connected. If the chemical potential of the capsule is lower than that of the reservoir, the capsule is the anode of the battery. In the opposite case it constitutes the cathode of the stack.

The present invention also relates to a method for producing an anode and / or a bacterial cell cathode, comprising the implementation of a capsule according to the invention.

The present invention also relates to an anode and / or a bacterial cell cathode comprising a capsule according to the invention.

More particularly, said anode and / or cathode further comprises a reservoir as mentioned above, the capsule and the reservoir being electrically connected and separated by an ion exchange membrane.

According to one of these aspects, the present invention also relates to a method for screening electroactive bacteria comprising culturing bacteria contained in capsules according to the invention as defined above.

As demonstrated in the experimental part, it is possible to encapsulate one bacterium per capsule to create encapsulated monoclonal cultures. It is also possible to measure the production of electric current generated by a population of bacteria encapsulated in a conductive membrane capsule. The encapsulation method allows the creation of capsules in series.

The screening consists of varying a parameter and selecting a value of this parameter that satisfies a predefined criterion. The selection of the value of the parameter is generally done by a measurement corresponding to the criterion defined.

In the context of the present invention, the parameter: "electroactive bacteria" varies according to the criterion: "electrical performance".

According to one of these aspects, a method for screening electroactive bacteria according to the invention comprises:

at. encapsulation of electroactive bacteria in the capsules according to the invention, each capsule comprising an electroactive bacterium;

b. obtaining monoclonal colonies of electroactive bacteria;

vs. the electrical connection of said capsules to a potentiostat; and

d. measuring the current output for each capsule.

In particular, the monoclonal colonies of electroactive bacteria of step b. result from the division of said electroactive bacteria in each capsule.

In particular, during the electrical connection of step c, the capsules are placed at a potential of 0.4 V relative to an Ag _(s) / AgCl _(s) electrode in the presence of a counter electrode. In particular, the screening method according to the invention comprises an additional step e. in which the best performing monoclonal colony in electrical production is selected.

The culture conditions of the electroactive bacteria are determined by those skilled in the art according to each bacterium on the basis of his general knowledge.

In the context of the present invention, the bacteria are cultured in a culture medium comprising a basic culture medium, the latter having the characteristic of being able to support the growth and development of said bacteria, in particular for the obtaining a monoclonal line. This type of basic culture medium is well known to those skilled in the art. There may be mentioned, for example, LB Broth medium or 822 medium of DMSZ.

In the context of the present invention, when the capsule must comprise a monoclonal population of bacteria, the skilled person can use the Poisson's law to determine the concentration of bacteria to be encapsulated to obtain a bacterium per capsule: p (k) = P (X = k) =

, in which :

- λ is the average number of bacteria per capsule volume; and

- P (k) is the probability that there are k bacteria in a capsule knowing the value of λ.

The value of λ depends on the concentration C of the cells of the first solution, expressed as the number of cells per unit volume, the diameter D of the capsule and the ratio r _q of the flow rates, as follows: λ = D ³ xr _q xC / 6.

According to another of these aspects, the present invention also relates to a device comprising at least one capsule according to the invention and at least one electrode, said at least one electrode being in contact with the gelled envelope of said at least one capsule. The present invention will be further illustrated by the following figures and examples which do not limit the scope thereof.

FIGURES

Figure 1: Left: Formation of formation of the bead composed of A in the heart and B around. Right: Extremity of the injector allowing the formation of the bead drop. (A) Exit for the mixture A constituting the heart of the capsule, (B) outlet for the mixture B constituting the membrane of the future capsule.

Figure 2: Diagram of the manufacturing assembly of the liquid core capsules and conductive membrane (D = 3mm, η = 350μηι, R = 1, 7mm, H = 5cm)

Figure 3: Liquid heart and conductive membrane capsules

4: Evolution of the conductivity of a capsule according to the invention as a function of the mass percentage of nanotubes

Figure 5: Diagram of the assembly of encapsulation of bacteria with a concentration of the heart solution set to have a bacterium per capsule (D = 3mm, ή = 350μηι, R = 1, 7mm, H = 5cm)

Figure 6: Mounting of the connection device of the conductive membrane capsule and the electrode system for measuring the current extracted from the capsule with A = PDMS (polydimethylsiloxane) for the mechanical compression of the capsule, B = conductive membrane capsule connected, C = measuring electrode: platinum wire, D = counter electrode: platinum gate, E = reference electrode: Ag _(S) wire _/ AgCl _(S)

Figure 7: Mounting of the connection device of the conductive membrane capsule and the electrode system for measuring the current extracted from the capsule

Figure 8: Temporal evolution of the current from a conductive membrane capsule containing Geobacter sulfurreducens

EXAMPLES

Protocol for manufacturing liquid heart capsules and semi-permeable conductive membrane

Creation of the gélifiable mixture:

The mixture: 10 mL of milliQ water, 150 mg of carbon nanotubes and 200 mg of SDS is dispersed under ultrasound for 1 h 15 with a Vibracell 75042 Bioblock Scientific sonicator according to the following parameters: Power = 35% Pmax; Puise: 3s on, 1 s off. The alginate powder LF200FTS, 1% by weight, is then added to the dispersion and then the whole is stirred magnetically for 12 hours in order to obtain the gelling solution.

Creation of the mixture to encapsulate:

The mixture: 25 mL of milliQ water, 5.1 g of polyethylene glycol of average molecular weight 3350 (PEG 3350 CAS 25322-68-3 Sigma Aldrich) and 135 mg of NaCl are mixed with magnetic stirring for 3 h.

Creation of the gelation bath:

1 l of milliQ water, 10 g of CaCl 2, 50 μl of tween 20 at 10% by weight are mixed with magnetic stirring for 3 h. Creation of the capsule:

A two-drop is created with the device shown in Figures 1 and 2 according to the method described in WO2010 / 063937.

The mixture to be encapsulated is placed in the syringe A, it will leave the center of the injector (A) to form the heart of the future capsule.

The gélifiable mixture is placed in the syringe B, it will leave the edge of the injector (B) to form the membrane of the future capsule.

The creation of the dual-dropper and its parameters are controlled by controlling the flow rates of the two solutions (heart and exterior) via syringe shoots. The flow rate of the syringe A is 5 ml / h, the flow rate of the syringe B is 5 ml / h.

The bead drop is then gelled by dropping it into the gelling bath. The outer solution gels (alginate gels) and encloses the liquid heart of the bead. The capsule is created as shown in Figure 3. The capsule is left in the gel bath for 1 h.

Description of the capsule:

The flow rates for capsule formation are 5 ml / h for the outer solution (membrane) and 5 ml / h for the inner solution (core).

The injector has a diameter of 3mm (D), it is placed at 5cm above the gelation bath (H).

For these flow rates, the final capsule has a diameter of 1, 7mm (R), with a membrane of 350μηι average thickness (h). The heart of the capsule has a volume of 1 1 μ.

Characterization of the conductivity of the gélifiable mixture

The evolution of the conductivity of the gélifiable mixture is measured using an impedance meter (Materials Mate 7260) on solid gel beads at 10 kHz, as a function of the mass percentage of nanotubes.

Creation of gelifiable mixtures:

The protocol for creating mixtures with different mass percentages of carbon nanotubes is the same as that of the protocol for creating the gelling mixture of the creation of the capsules. Only the mass of nanotubes changes. 5 different mixtures are made with the following masses of nanotubes: 0; 50; 100; 150; 200 mg corresponding respectively to the following percentages: 0; 0.5; 1; 1, 5; 2% by mass.

Creation of the gel beads:

Using a VWR plastic pasteur pipette of 7 ml, the gelling mixtures are dripped from a height of 5 cm into the gelling bath (identical to that described above). The beads are allowed to gel for 24 hours in the bath. The marbles are then equilibrated in a solution of CaCl 2 at 1 mM for 48 hours to dialyze the SDS present in the gélifiable mixture.

3 mm diameter solid balls composed of 1% by weight of alginate and mass percentages of variable carbon nanotubes are thus obtained.

Measurement protocol:

To measure the conductivity, a ball is placed between two screws of 3mm diameter 2.7mm apart, connected to the impedance meter. The conductivity at 10 kHz is measured.

The results are shown in Figure 4.

The results show an increase in the conductivity of the gel with the addition of the nanotubes. The addition of carbon nanotubes makes it possible to increase the conductivity of the hydrogel up to 100 times.

Protocol for manufacturing capsules comprising bacteria for monoclonal cultures.

For the encapsulation of bacteria, all the solutions used are sterilized by 0.2μηι filtration. The encapsulation device is thermally sterilized at 121 ° C. for 20 minutes by an autoclave (JSM table top sterilizer).

Creation of the gélifiable mixture:

1 .7% by weight of alginate LF200FTS and 5mM of SDS are dissolved in 10 ml of milliQ water and then stirred magnetically for 12 hours to obtain the gellable mixture.

Creation of the gelation bath:

1 l of milliQ water, 10 g of CaCl 2, 50 μl of tween 20 at 10% by weight are mixed with magnetic stirring for 3 h.

Monoclonal Encapsulation: Determination of the monoclonal concentration allowing to have one (or less) bacterium per capsule.

To prepare the bacteria solution at the monoclonal concentration, the Poisson's law is used: p (k) P (xk) _fc! e | _a q _U it:

- λ is the average number of bacteria per capsule volume;

- P (k) is the probability that there are k bacteria in a capsule knowing the value of λ; and

the volume of the heart of a capsule is 1 1 μ.

Thus, by applying this law with 0.1 bacteria per capsule (9 bacteria / m L), 90% of the capsules are without bacteria (p (0)), 9% of the capsules obtained are monoclonal, it is up to say with a bacterium (p (1)), less than 0.5% of the capsules obtained are with two bacteria (p (2)) and less 0.02% of the capsules obtained are with 3 bacteria, etc.

This protocol thus makes it possible to obtain monoclonal capsules with a low risk of having capsules containing bacteria resulting from a non-monoclonal encapsulation (5% of the capsules containing bacteria).

Creation of the bacterial mixture to encapsulate:

The mixture: 8.3 ml of milliQ water, 1.7 g of polyethylene glycol of average molecular weight 3500 (PEG 3350 CAS 25322-68-3 Sigma Aldrich) and 45 mg of NaCl are mixed with magnetic stirring for 3 h. Then 1 ml of bacterial solution consisting of the bacteria is added to the concentration of 84 bacteria per milliliter in its culture medium (see below).

Thus, a bacterial solution comprising E. coli bacteria is prepared as follows: 2 μL of an E. coli strain MC4100 YFP stored at -80 ° C. is added to 10 ml of LB Broth (Sigma). The mixture is cultured with circular stirring for 12 hours at 37 ° C. At 12h, the bacterial concentration is evaluated by an absorbance measurement at 600 nm. The difference in optical density observed at 600nm between the LB Broth medium and the bacterial culture (Delta.DO in optical density unit) makes it possible to obtain the concentration of bacteria per milliliter via the formula: Concentration = Delta.DO ^* 4.10 ⁸ bacteria / mL. This culture is then diluted by successive dilutions in LB Broth to reach the concentration of 84 bacteria / ml.

Thus, a bacterial solution comprising Geobacter bacteria is prepared in the following manner: 1 ml of a strain of bacteria Geobacter Sulfurreducens supplied by DMSZ is added to 9 ml of its specific medium (826 DMSZ medium) in a hungate tube of 12 ml under anaerobic at 30 ° C for 4 days. At 4 days, the bacteria concentration is evaluated by an absorbance measurement at 600 nm. The difference in optical density observed at 600nm between the culture medium 826 and the bacterial culture (Delta.DO in optical density unit) makes it possible to obtain the concentration of bacteria per milliliter via the formula: Concentration = Delta.DO ^* 4.10 ⁸ bacteria / mL. This culture is then diluted by successive dilutions in medium 826 to reach the concentration of 84 bacteria / ml.

Creation of the monoclonal culture capsule:

A two-drop is created with the device shown in Figures 1 and 5 according to the method described in WO2010 / 063937.

The bacterial mixture to be encapsulated (prepared with E. coli or Geobacter bacteria as indicated in the previous paragraph) is placed in the syringe A, it will leave the center of the injector (A) to form the heart of the future capsule . The gélifiable mixture is placed in the syringe B, it will leave the edge of the injector (B) to form the membrane of the future capsule.

The bead drop is then gelled by dropping it into the gelling bath. The outer solution gels (alginate gels) and encloses the liquid heart of the bead. The capsule is created. The capsule is left in the gel bath for 15min. The capsules are then cultured in the culture medium of the encapsulated bacterium (cf previous paragraph) supplemented with 5 mM of Cacl2, and under the culture conditions applicable to the encapsulated bacterium. After the incubation, capsules filled with bacteria are obtained.

The results of the monoclonal encapsulation are shown in Table 1 below.

Table 1:

Protocol for the manufacture of Geobacter-enclosed semi-permeable conductive membrane liquid-core capsules and connection device for the recovery of electrons produced by encapsulated bacteria

Creation of the gélifiable mixture

The mixture: 10 mL of milliQ water, 200 mg of carbon nanotubes and 150 mg of Brij35 is dispersed under ultrasound for 1 h 15 with a Vibracell 75042 Bioblock Scientific sonicator according to the following parameters: Power = 35% Pmax; Puise: 3s on, 1 s off. The alginate powder LF200FTS, 1.5% by weight, is then added to the dispersion and then everything is stirred magnetically for 12 hours. 1 mM SDS is added with magnetic stirring to obtain the gelling solution.

Creation of the encapsulating mixture containing Geobacter Sulfurreducens

On the one hand the following mixture is prepared: 25 ml of milliQ water, 5.1 g of polyethylene glycol of average molar mass 3350 (PEG 3350 CAS 25322-68-3 Sigma Aldrich) and 135 mg of NaCl are mixed with magnetic stirring during 3h.

On the other hand, a bacterial solution comprising Geobacter bacteria is prepared as follows:

1 ml of a strain of Geobacter Sulfurreducens bacteria supplied by DMSZ is added to 9 ml of its specific medium (826 DMSZ medium) in a 12 ml hungate tube under anaerobic conditions at 30 ° C. for 4 days. At 4 days, the bacteria concentration is evaluated by an absorbance measurement at 600 nm. The difference in optical density observed at 600nm between medium 826 and the bacterial culture (Delta.DO in optical density unit) gives the bacterial concentration per milliliter via the formula: Concentration = Delta.DO ^* 4.108 Bac / ml. This culture of Geobacter is diluted to 20 ^th in this solution. The bacterial mixture to be encapsulated is then obtained.

Creation of the qélification bath

1 L of milliQ water, 10 g of CaCl 2, 50 μl of tween 20 to 10% by weight are mixed with magnetic stirring for 3 hours. Creation of the capsule

A drip is created with the device shown in Figures 1 and 2.

The creation of the dual-dropper and its parameters are controlled by controlling the flow rates of the two solutions (heart and exterior) via syringe shoots. The flow rate of the syringe A is 5 ml / h, the flow rate of the syringe B is 4 ml / h.

The bead drop is then gelled by dropping it into the gelling bath. The outer solution gels (alginate gels) and encloses the liquid heart of the bead. The capsule is created as shown in Figure 2. The capsule is left in the gel bath for 3 minutes.

The capsule is then transferred to the specific medium of Geobacter. The culture is kept for 4 days under anaerobic at 25 degrees Celsius. The medium is not buffered with surfactants used in the formulation of the gelling mixture. By dialysis, the surfactant is desorbed from the surface of the nanotubes. Unstabilized, the nanotubes connect to each other and create a conductive network within the hydrogel membrane.

Device for the electrical connection of the capsule

The device consists of three electrodes: a reference electrode silver chloride (Ag (s) / AgCl (s)), a counter electrode composed of a platinum grid and a measuring electrode composed of a wire of platinum. These three electrodes are connected to a potentiostat which allows them to be put under potential. The measuring electrode allows the connection of a capsule by a slight physical compression as illustrated in Figure 5 and Figure 6. The three electrodes are immersed in 826 DMSZ medium without sodium fumarate.

Capsule connection and current recovery

After 4 days, the capsule is positioned in the connection device described above, in an anaerobic environment. The capsule is put under potential at 0.4V. The current is followed by chronoamperometry. Figure 7 shows the evolution of the current as a function of time without capsule (approximately 4 ^* 10-8 A), then after the addition of a conductive membrane capsule containing Geobacter in the connection device.

The current reaches a value of 1, 7 ^* 10 ^"5 A.

At the end of the experiment, the capsule is destroyed in Geobacter specific medium (826 DMSZ medium). The encapsulated bacteria are thus recovered. The concentration of bacteria is estimated by an optical density measurement.

The concentration of bacteria inside the capsule is of the order of 6.10 ⁹ bac / mL.

Thus, the capsule according to the invention has many advantages:

Parallelization possible and not limited;

- Growth at long time; Simplified recovery of the entire cultivated population (no centrifugation);

Possible encapsulation of a single microorganism;

Possibility of electrical type actions on the encapsulated organism;

- Simplified manipulation of the culture; and

- bioreactor dissociated from the current measurement system.

Claims

1. Capsule comprising a liquid core, a gelled envelope completely encapsulating the liquid core at its periphery, the gelled envelope being adapted to retain the liquid heart when the capsule is immersed in a gas, an aqueous solution or an oil, the gelled envelope comprising at least one gelled polyelectrolyte and at least one surfactant, characterized in that the gelled envelope further comprises an electrically conductive material.

2. Capsule according to claim 1, characterized in that it is obtained by the implementation of a method comprising the following steps of:

e) separated conveying in a double jacket of a first aqueous or oily liquid solution and a second liquid solution containing a liquid polyelectrolyte suitable for gelling and the electrically conductive material;

f) forming, at the exit of the jacket, a series of drops, each drop comprising a central core formed of said first solution and a peripheral film formed of said second solution and completely covering the central core;

g) immersing each drop in a gelling solution containing a reagent capable of reacting with the polyelectrolyte of the film to change it from a liquid state to a gelled state and forming the gelled envelope comprising the electrically conductive material, the central core forming the liquid heart;

h) recovery of formed capsules;

The capsule according to claim 2, wherein said second liquid solution containing a clean liquid polyelectrolyte to be gelated and the conductive material mentioned in step a) of the process is formed by contacting the electrically conductive material in dispersed form and the polyelectrolyte clean to gel.

4. Capsule according to any one of claims 1 to 3, characterized in that it further comprises an intermediate envelope encapsulating completely to its periphery the liquid core, said intermediate envelope itself being completely encapsulated at its periphery by the gelled envelope.

5. Capsule according to any one of claims 1 to 4, wherein the liquid core comprises at least one electro-active object.

6. Capsule according to any one of the preceding claims, wherein the electrically conductive material consists of electrically conductive solid particles.

7. Capsule according to claim 6, wherein said solid electrically conductive particles are selected from carbon nanotubes, carbon black, graphene.

8. Capsule according to any one of claims 6 or 7, wherein the gelled envelope is formed by a divalent ion gelled polysaccharide hydrogel comprising said solid electrically conductive particles in dispersed and connected form thereby forming an electrically conductive network.

The capsule of any preceding claim, wherein said electrically conductive material is biocompatible.

The capsule of claim 5, wherein said electroactive object is selected from microorganisms, cells, macromolecules and electroactive solid particles.

1 1. The capsule of claim 10, wherein said microorganism is selected from bacteria of the genus Escherichia coli, Geobacter, Shewanella, Rhodopseudomonas, Ochrobactrum, Enterobacter.

12. Use of at least one capsule according to any one of claims 1 to 1 1, for the production of bioreactors.

13. Use of at least one capsule according to any one of claims 10 to 1 1, for the screening of microorganisms, in particular electro-active microorganisms.

14. Use of at least one capsule according to any one of claims 10 to 1 1, for the recovery of electrons produced by microorganisms.

15. Use of at least one capsule according to any one of claims 10 to 1 1, for the production of an anode and / or a bacterial cell cathode.

16. A method of screening for electroactive bacteria comprising culturing bacteria contained in capsules as defined in any one of claims 1 to 11 by:

at. encapsulating electroactive bacteria in the capsules according to any one of claims 1 to 1 1, each capsule comprising an electroactive bacterium;

b. obtaining monoclonal colonies of electroactive bacteria;

vs. the electrical connection of said capsules to a potentiostat; and

d. measuring the current output for each capsule.

17. Device comprising at least one capsule according to any one of claims 1 to 1 1 and at least one electrode, said at least one electrode being in contact with the gelled envelope of said at least one capsule.