FR3147814A1 - NUTRIENT MEDIUM FOR BIOLUMINESCENT BACTERIA AND DEVICE FOR ITS IMPLEMENTATION - Google Patents

Abstract

The invention relates to the field of bioluminescent devices. More particularly, the invention relates to a nutrient medium optimized to promote the intensity and duration of bioluminescence produced by non-genetically modified marine bacteria, in particular photobacteria of the species Photobacterium kishitanii (in particular the strain deposited with the CNCM on March 16, 2023 under number CNCM I-5938) and Photobacterium phosphoreum. The invention also relates to a light device for the optimal implementation of the nutrient medium and photobacteria.

Description

NUTRIENT MEDIUM FOR BIOLUMINESCENT BACTERIA AND DEVICE FOR ITS IMPLEMENTATION

The invention relates to the field of bioluminescent devices. More particularly, the invention relates to a nutrient medium optimized to promote the intensity and duration of bioluminescence produced by non-genetically modified marine bacteria, in particular photobacteria of the species Photobacterium kishitanii (in particular the strain deposited with the CNCM on March 16, 2023 under the number CNCM I-5938) and Photobacterium phosphoreum . The invention also relates to a light device for the implementation of the nutrient medium and the photobacteria in an optimal manner.

Field of invention

Bioluminescence is a phenomenon of light production by living organisms. Bioluminescent bacteria are mainly present in the marine environment in different forms: isolated bacteria (floating), biofilms or in symbiosis with a multitude of marine organisms such as fish, cephalopods and cnidarians.

The most studied bioluminescent bacterium is Allivibrio fisheri . But other bioluminescent bacteria have been found among the genera Vibrio , Photobacterium , Aliivibrio , Photorhabdus and Shewanella .

The mechanism of light production by bioluminescent bacteria involves a chain of enzymatic reactions catalyzed by proteins produced by genes of the luxCDABEG operon. The molecules transformed by these enzymes emit light at a wavelength of 490 nm, which corresponds to a blue-green color. The intensity of light produced by photobacteria depends on the level of expression of the luciferase LuxAB as well as other genes of the lux operon. It may also be dependent on the level of expression of genes responsible for the production of its substrate: reduced flavin mononucleotide (FMNH ₂ ) as well as long-chain aldehydes that participate in the light production reaction.

One of the essential factors for the production of bioluminescence is the supply of oxygen via aeration of the bacterial culture. Oxygen serves as an electron acceptor and allows the emission of photons during the enzymatic reaction catalyzed by luciferase, a key enzyme in the production of light. In the absence of oxygen, the production of bioluminescence by the bacterial culture ceases. It will only resume when aeration and therefore the supply of oxygen are effective again.

Other factors controlling bioluminescence are described. For example, the lux operon is known to be repressed by the presence of glucose. Quorum sensing is another mechanism controlling the expression of the lux operon in the bioluminescent bacterium Vibrio fischeri . This bacterium has the ability to detect the density of its population and activate the expression of the lux operon and the production of light when the cell density reaches a sufficiently high level. However, this mechanism is not effective in the species Photobacterium phosphoreum or Photobacterium kishitanii and the light produced by these bacteria is directly proportional to the cell density.

Bioluminescence arouses curiosity and interest as a source of liquid and biological lighting. Improving the production performance of this light helps to broaden its fields of application.

Disadvantages of the state of the art

Without considering the widespread use of bioluminescence in genetically modified systems, the use of natural (wild) bioluminescent bacteria is limited to water contamination tests (e.g. BioFix Lumi kits from Macherey Nagel and Microtox from Phytocontrol), to the production of light as part of an illumination system for a complex decorative device (patent WO2018/087504 A1), for educational purposes (bioluminescence kits from Leuchtlabor, Germany), for events at private homes (patent FR3122184A1) or for the creation of innovative play products (patent WO97/29319). The growth medium generally used is a standard medium for marine bacteria such as Marine Broth 2216 or Photobacterium Broth. However, despite good growth in these environments, bioluminescent bacteria emit light with a fairly low level of intensity and duration of expression, which limits its use.

The inventors have developed a nutrient medium allowing a significant improvement in the intensity and duration of light production by bioluminescent bacteria. This medium is suitable for photobacteria of the species Photobacterium kishitanii and Photobacterium phosphoreum , in particular the strain of Photobacterium kishitanii deposited with the CNCM on March 16, 2023 under number CNCM I-5938.

• Au moins une source d’azotée choisie parmi un extrait de levure ou de la peptone de levure, la concentration totale en ladite source azotée étant d’au moins 1 g /L (0,1%) ;
• Au moins une source carbonée choisie parmi (i) du glycérol ou (ii) le gluconate de sodium, la concentration totale en ladite source carbonée étant d’au moins 3 g/L (0,3%) ;
• Un sel choisi parmi (i) le NaCl à une concentration d’au moins 20 g/L (2%), lorsque ladite source carbonée est du glycérol ou (ii) le NaCl à une concentration d’au moins 20 g/L (2%), ou le KCl à une concentration d’au moins 20 g/L (2%), ou du sel de mer à une concentration d’au moins 38 g/L (3,8%), lorsque ladite source carbonée est le gluconate de sodium

Thus the invention relates to a nutrient medium for bioluminescent bacteria comprising:

• At least one nitrogen source selected from a yeast extract or yeast peptone, the total concentration of said nitrogen source being at least 1 g/L (0.1%);
• At least one carbon source selected from (i) glycerol or (ii) sodium gluconate, the total concentration of said carbon source being at least 3 g/L (0.3%);
• A salt selected from (i) NaCl at a concentration of at least 20 g/L (2%), when said carbon source is glycerol or (ii) NaCl at a concentration of at least 20 g/L (2%), or KCl at a concentration of at least 20 g/L (2%), or sea salt at a concentration of at least 38 g/L (3.8%), when said carbon source is sodium gluconate.

as well as its use to cultivate photobacteria and optimize their light production.

The invention also relates to a bioluminescent device including a culture vessel comprising an oxygenation system and a nutrient medium optimized to increase the luminous performance and bioluminescent bacteria belonging to the species Photobacterium kishitanii and Photobacterium phosphoreum .

Thus, the invention relates to a luminous device comprising a container with a height of at least ten centimeters comprising at least one transparent wall and having an oxygenation system, a nutrient medium as defined above and at least one bioluminescent bacterium chosen from a bacterium of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum .

• la souchePhotobacterium kishitaniidéposée auprès de la CNCM le 16 mars 2023 sous le numéro CNCM I-5938, et
• un kit pour la mise en œuvre d’un procédé de production de bioluminescence
• un procédé de production de bioluminescence.

The invention also relates to:

• the strain Photobacterium kishitanii deposited with the CNCM on March 16, 2023 under number CNCM I-5938, and
• a kit for the implementation of a bioluminescence production process
• a process for producing bioluminescence.

Advantages of the invention

The present invention provides a solution to overcome the limitations of using photobacteria for implementing a bioluminescent device to create ambient illumination by proposing an innovative nutrient medium. This nutrient medium was developed to optimize the intensity and duration of bioluminescence and simplify its preparation. It is based on an optimized formulation with regard to the choice of carbon, nitrogen and salt sources necessary for the proper growth and maintenance of the bacterial culture as well as the production of bioluminescence.

This medium was developed specifically for non-genetically modified photobacteria, so that bacterial cultures can be used for the production of bioluminescence, without the restrictions associated with GMO regulatory provisions.

The nutrient medium is intended for use in a device that does not benefit from an external supply of nutrients or bacteria. Only an oxygen supply is required; this can be obtained by a bubbling system (or equivalent) or simple agitation without a bubbling system.

This bioluminescent device is therefore based on the culture of bioluminescent bacteria in a liquid medium. It stands out from existing bioluminescent devices by a more intense light performance greater than 3 lux and an increased duration of several hours at 22°C (at least 12 hours according to prototyping) without any other nutrient supply during culture.

The device can emit bioluminescence for up to about 12 hours, which defines its usage time. Due to its ease of use, it can be used in outdoor and indoor spaces, including private homes.

DETAILED DESCRIPTION OF THE INVENTION

• Au moins une source d’azotée choisie parmi un extrait de levure ou de la peptone de levure, la concentration totale en source azotée étant d’au moins 1 g /L (0,1%), de préférence étant comprise entre 1 g à 30 g/L (0,1 à 3%) ;
• Au moins une source carbonée choisie parmi (i) du glycérol ou (ii) le gluconate de sodium, la concentration totale en source carbonée étant d’au moins 3 g/L (0,3%), de préférence comprise entre 3 g à 12 g/L (0,3 à 1,2%) ;
• Un sel choisi parmi (i) le NaCl à une concentration d’au moins 20 g/L (2%), de préférence comprise entre 20 à 40 g/L (de 2 à 4%) lorsque la source carbonée est du glycérol ou (ii) le NaCl à une concentration d’au moins 20 g/L (2%), de préférence comprise entre 20 à 40 g/L (de 2 à 4%) ou le KCl à une concentration d’au moins 20 g/L (2%), de préférence comprise entre 20 à 30 g/L (de 2 à 3%) ou du sel de mer à une concentration d’au moins 38 g/L (3,8%), de préférence comprise entre 38 à 40 g/L (de 3,8 à 4%) lorsque la source carbonée est le gluconate de sodium.

A first object of the invention relates to a nutrient medium for bioluminescent bacteria comprising:

• At least one nitrogen source selected from a yeast extract or yeast peptone, the total concentration of nitrogen source being at least 1 g/L (0.1%), preferably being between 1 g to 30 g/L (0.1 to 3%);
• At least one carbon source selected from (i) glycerol or (ii) sodium gluconate, the total concentration of carbon source being at least 3 g/L (0.3%), preferably between 3 g to 12 g/L (0.3 to 1.2%);
• A salt selected from (i) NaCl at a concentration of at least 20 g/L (2%), preferably between 20 and 40 g/L (2 to 4%) when the carbon source is glycerol or (ii) NaCl at a concentration of at least 20 g/L (2%), preferably between 20 and 40 g/L (2 to 4%) or KCl at a concentration of at least 20 g/L (2%), preferably between 20 and 30 g/L (2 to 3%) or sea salt at a concentration of at least 38 g/L (3.8%), preferably between 38 and 40 g/L (3.8 to 4%) when the carbon source is sodium gluconate.

In particular embodiments of the invention, the amount of carbon source is adjusted according to the nature of said source, namely preferably between 3 and 10 g/L for glycerol and between 6 and 12 g/L for sodium gluconate.

• Au moins une source d’azotée choisie parmi un extrait de levure ou de la peptone de levure, la concentration totale en source azotée étant comprise entre 3 et 12 g/L ;
• Au moins une source carbonée choisie parmi (i) du glycérol à une concentration comprise entre 3 et 10 g/L ou (ii) le gluconate de sodium à une concentration comprise entre 6 et 12 g/L ;
• Un sel choisi parmi (i) le NaCl à une concentration comprise entre 20 et 40 g/L lorsque la source carbonée est du glycérol ou (ii) le NaCl à une concentration comprise entre 20 et 40 g/L ou le KCl à une concentration comprise entre 20 et 30 g/L ou du sel de mer à une concentration comprise entre 38 et 40 g/L lorsque la source carbonée est le gluconate de sodium.

In a preferred embodiment of the invention, the nutrient medium comprises:

• At least one nitrogen source selected from a yeast extract or yeast peptone, the total concentration of nitrogen source being between 3 and 12 g/L;
• At least one carbon source selected from (i) glycerol at a concentration of between 3 and 10 g/L or (ii) sodium gluconate at a concentration of between 6 and 12 g/L;
• A salt selected from (i) NaCl at a concentration of between 20 and 40 g/L when the carbon source is glycerol or (ii) NaCl at a concentration of between 20 and 40 g/L or KCl at a concentration of between 20 and 30 g/L or sea salt at a concentration of between 38 and 40 g/L when the carbon source is sodium gluconate.

In particular embodiments of the invention, the nutrient medium according to the invention is a medium comprising a carbon source chosen from a yeast extract and a yeast peptone and (i) the salt is sodium chloride and the nitrogen source is sodium gluconate (medium 1), or (ii) the salt is potassium chloride and the nitrogen source is sodium gluconate (medium 2), or (iii) the salt is sea salt and the nitrogen source is sodium gluconate (medium 3) or (iv) the salt is sodium chloride and the nitrogen source is glycerol (medium 4).

The nutrient medium may be in powder form, except for the glycerol which is in liquid form (the medium is then provided partly in powder form and partly in liquid form, in two separate containers; the ingredients are then mixed to reconstitute the liquid medium). The dehydrated form is particularly suitable for providing the medium and reconstituting it in liquid form in a bioluminescent device according to the invention. When glycerol is present in the nutrient medium, it can be provided for example using a syringe which facilitates its dosage. Whether the medium is only in powder form (when the carbon source is in the form of sodium gluconate), or in powder form for the nitrogen sources and salt and in liquid form for the carbon source (when the latter is glycerol), the nutrient medium will be reconstituted in the same way by mixing the ingredients in water.

Thus, for the purposes of the invention, the term “nutrient medium in powder form” is opposed to “ready-to-use liquid nutrient medium” and includes the complete powder form and the powder form with the addition of glycerol.

A second subject of the invention relates to the use of a nutrient medium as described above for cultivating photobacteria of the species Photobacterium kishitanii or Photobacterium phosphoreum .

These bacteria are natural bacteria, that is to say not genetically modified, also called wild bacteria.

In a preferred embodiment of the invention, the bacterium is a strain of Photobacterium kishitanii deposited with the CNCM on March 16, 2023 under the number CNCM I-5938

A third object of the invention concerns the strain of Photobacterium kishitaniifiled with the CNCM on March 16, 2023 under number CNCM I-5938 as such.

This strain has never been described before. It is a bioluminescent bacterium whose light production performance is improved thanks to the nutrient medium according to the invention.

A fourth subject of the invention relates to a luminous device comprising a container with a height of at least ten centimeters comprising at least one transparent wall and having an oxygenation system, a nutrient medium as described above and a bioluminescent bacterium chosen from a bacterium of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum.

The volume of the container is not a constraint. For example, it can contain from 50 ml to 50 L and more by adapting the air supply parameters. The shape of the container and its size can be variable, but will preferably be vertical to promote oxygenation. The diameter will preferably be at least 5 cm in diameter. The container must have free openings in its upper part to allow the evacuation of the injected air.

The container can be completely or partially transparent depending on the desired effect.

By “oxygenation system” is meant any system allowing the supply of oxygen into the nutrient medium. This supply of oxygen is preferably obtained by introducing air into the nutrient medium, this passage of air in the form of bubbles in the medium ensuring a dissolution of oxygen then available for the bioluminescent bacteria. Any bubbling system is therefore suitable, whatever the size of the bubbles. The air can be introduced into the medium via a perforation in the wall of the container, a pipe, nozzles or any other equivalent means. This oxygenation system can also consist of a system allowing sufficient agitation of the medium to supply oxygen.

In a fifth object, the invention may be provided in the form of a kit comprising a container comprising an oxygenation system, a nutrient medium in powder form and bioluminescent bacteria of the speciesPhotobacterium kishitaniior of the speciesPhotobacterium phosphorusin freeze-dried form.

The nutrient medium in the kit (medium to be reconstituted) can only be in powder form when the carbon source is provided by sodium gluconate. When the carbon source is provided by glycerol, it is present in the kit in an additional container due to its liquid form; this does not change the principle of the kit, since it is sufficient to mix the ingredients (powder + glycerol) with water to reconstitute a ready-to-use nutrient medium. This is explained above.

• Mettre dans un récipient d’une hauteur d’au moins une dizaine de centimètres comportant un système d’oxygénation de l’eau et un milieu nutritif tel que décrit précédemment sous forme de poudre afin de reconstituer un milieu nutritif liquide ;
• Ajouter les bactéries bioluminescentes de l’espècePhotobacterium kishitaniiou de l’espècePhotobacterium phosphoreumsous forme lyophilisée
• Activer ledit système d’oxygénation
• Cultiver ainsi les bactéries pendant au moins 10h pour voir apparaître la lumière.
• Poursuivre la culture pendant la durée voulue ou jusqu’à extension de la bioluminescence.

A sixth subject of the invention relates to a method for implementing bioluminescence comprising the following steps:

• Place in a container at least ten centimeters high with a water oxygenation system and a nutrient medium as described above in powder form in order to reconstitute a liquid nutrient medium;
• Add bioluminescent bacteria of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum in freeze-dried form
• Activate said oxygenation system
• Cultivate the bacteria for at least 10 hours to see the light appear.
• Continue culture for desired duration or until bioluminescence extends.

The medium can be reconstituted by adding water, particularly tap water, but any type of water will do.

The present invention will be better understood from the following examples, provided for illustration purposes and in no way to be considered as limiting the scope of the present invention.

DESCRIPTION OF FIGURES

: Comparison of the luminous performance of the media with the claimed carbon sources.

[Fig.2][Fig.2] : A - Bioluminescence production kinetics in medium with glycerol and a range of NaCl concentrations. B - Bioluminescence production kinetics in medium with sodium gluconate and a range of NaCl concentrations.

[Fig.3][Fig.3] : A - Kinetics of bioluminescence production in a medium containing sodium gluconate and a range of KCl concentrations. B - Kinetics of bioluminescence production in a medium containing glycerol and a range of KCl concentrations.

: A - Kinetics of bioluminescence production in a medium containing sodium gluconate and a range of sea salt concentrations. B - Kinetics of bioluminescence production in a medium with glycerol and a range of sea salt concentrations.

: Comparison of the light performance of photobacteria cultivated either in the nutrient medium according to the invention or in a commercial medium.

EXAMPLES

The inventors have carried out exhaustive tests to define the components of the medium and their optimal concentrations to promote the expression of bioluminescence. Typically the nutrient medium for bacteria is composed of a carbon source (sugars and their derivatives), a nitrogen source (proteins, peptides, amino acids), vitamins and mineral salts.

EXAMPLE 1: Materials and methods Development of the culture medium

Bacteria are available in lyophilized form. The reculture conditions for microplate analysis are as follows: Corning Costar@Assay Black with Clear Flat Bottom 96-well microplates (Reference 3904) contain 200 µl of culture per well. They are inoculated with a preculture at an optical density (OD) of 0.1 at launch.

For comparative experiments, each well contains determined concentrations for all ingredients except the one tested which is added at varying concentrations. Growth and bioluminescence level are monitored over 24-60 hours in a Tecan Te-Cool Spark® microplate reader with temperature control (22°C). Bioluminescence data are presented in non-standardized relative light units (RLU).

The conditions used for the analysis presented on the consist of inoculating 50 ml of medium at OD 0.1 at launch. The inoculated media are shaken at 150 rpm in 250 ml baffled bottom Erlenmeyer flasks in an incubator at a temperature of 22 °C for 22 hours. Bioluminescence data are collected by an electronic device with light sensors calibrated with a lux meter (Konica Minolta T-10A model). A group of several sensors is connected to a microcontroller that times and sends the data to a server. Thus the light intensity emitted by the cultures is monitored for the desired time, typically over 24-72 hours.

Device implemented for bacterial culture

The medium described above is used to cultivate bioluminescent bacteria in a container that allows the oxygenation of the culture, oxygen being necessary for the emission of light. The container must be transparent, and can be of variable size and shape with a sufficient height of at least ten cm. It must be supplied by air bubbling or any other equivalent device that allows the mixing and oxygenation of the bacterial culture. It must also be equipped with free openings in its upper part to allow the evacuation of the injected air.

In a particular embodiment of the invention, the container is cylindrical with a height of 100 cm and a diameter of 5-20 cm (pipe). The injection of filtered air is done through the base of the cylinder using a compressor or an air pump. In another embodiment, the container is flat to serve as a display panel, 1-3 cm thick, 500 to 1000 mm high and of variable width. It must have air injection points in its lower part. The container can also be a glass bottle or can closed by a screw cap equipped with air supply pipes. The device can be used without air supply (bubbling) in different containers (tube, bottles, Erlenmeyer flasks) subjected to mechanical stirring (internal stirring device or stirring plate).

EXAMPLE 2 : Determination of s source s of carbon and salts in the culture medium to optimize bioluminescence

Choice of carbon source

The lux operon is known to be repressed by the presence of glucose, a mechanism called "catabolite repression." The inventors confirmed this repression by using glucose as the primary carbon source. The level of light produced by the bacteria in the presence of glucose is comparable to that produced in the absence of an additional carbon source ( ).

In order to select the best carbon source to promote the production of light by photobacteria, the inventors carried out a comparative study including a broad spectrum of sugars and its derivatives. These experiments were carried out in a microplate reader (Materials and Methods section). The shows the light production as a function of different carbon sources present in the culture medium. It reports a very significant increase in the light intensity emitted by the bacteria in the presence of two best carbon sources that the inventors selected: glycerol and sodium gluconate, compared to that observed in the presence of medium without an additional carbon source (as well as in the presence of glucose, as previously described).

Determination of the nature and quantity of salts

The natural environment of marine bacteria being seawater, it is essential to maintain a high osmotic level of its growth medium (as an indication, the concentration of all salts in seawater is between 30 and 40 g/L). Since the concentration of salts directly impacts bioluminescence, the inventors carried out growth and light production tests in microplates with concentration ranges for different salts: sea salt, NaCl, and KCl. Ranges of each of these 3 salts were tested successively with each of the two selected carbon sources.

The results obtained with NaCl are presented in [Fig.2]. They show that the optimal concentration of NaCl for light production is between 20 to 40 g/L, whether the carbon source is glycerol ( ) or gluconate ( ).

A similar analysis was carried out with potassium chloride and sea salt. The results obtained with potassium chloride are shown in [Fig.3]. The results obtained with sea salt are shown in .

They show that in the presence of glycerol, the level of bioluminescence obtained with KCl and sea salt is not satisfactory whereas in the presence of sodium gluconate, both salt sources can be used at a level of 20-30 g/L for KCL and 38-40 g/L for sea salt.

Choice of nitrogen source

The nitrogen sources selected by the inventors are preferably yeast extract or yeast peptone at a concentration of at least 1 g/L (0.1%), preferably between 1 g/L and 30 g/L.

Since yeast extract or yeast peptone contains the vitamins and microelements necessary for the growth of the bacterial culture, adding additional components is not useful because it does not increase the level of bioluminescence.

Examples of culture media according to the invention

The culture medium optimized according to the invention for the best light performance comprises the following components in the following quantities and weight proportions for 1L:

Medium 1: at least 1 g (0.1%), preferably 1 g to 30 g (0.1 to 3%) of a nitrogen source, preferably selected from a yeast extract and a yeast peptone, at least 20 g (2%), preferably 20 g to 40 g (2 to 4%) of sodium chloride (NaCl), at least 6 g (0.6%), preferably 6 g to 12 g (0.6 to 1.2%) of sodium gluconate

Medium 2: at least 1 g (0.1%), preferably 1 g to 30 g (0.1 to 3%) of a nitrogen source, preferably chosen from a yeast extract and a yeast peptone, at least 20 g (2%), preferably 20 g to 30 g (2 to 3%) of potassium chloride (KCl), at least 6 g (0.6%), preferably 6 g to 12 g (0.6 to 1.2%) of sodium gluconate

Medium 3: at least 1 g (0.1%), preferably 1 g to 30 g (0.1 to 3%) of a nitrogen source, preferably selected from a yeast extract and a yeast peptone, at least 38 g (3.8%), preferably 38 g to 40 g (3.8 to 4%) of sea salts, at least 6 g (0.6%), preferably 6 g to 12 g (0.6 to 1.2%) of sodium gluconate

Medium 4: at least 1 g (0.1%), preferably from 1 g to 30 g (0.1 to 3%) of a nitrogen source, preferably chosen between a yeast extract and a yeast peptone, at least 20 g (2%), preferably from 20 g to 40 g (2 to 4%) of NaCl, at least 3 g (0.3%), preferably from 3 g to 10 g (0.3 to 1%) of glycerol

These media are supplied in powder form and are taken up in tap water by the user.

E EXAMPLE 3 : Comparison between biolumin escence obtained with a standard nutrient medium and that obtained with the nutrient medium according to the invention

The bioluminescence produced by the bacteria cultured in the nutrient medium according to the invention was compared to that cultured in a commercial standard medium: it was established that the nutrient medium according to the invention allows the production of bioluminescence significantly higher in both intensity and duration. These results are presented in .

The light performances obtained by the nutrient medium according to the invention have the following characteristics: a light intensity greater than 3 lux for at least 10 hours for a 200 ml culture in 1 L baffle flasks shaken at 150 rpm and at 22°C or in the pipes supplied with air and those without any other nutrient supply during the culture. The bioluminescence peak greater than 3 lux corresponds to the OD between 2 and 5, the top of the peak being at OD 3.5-4.

EXAMPLE 4: Example of use of a device according to the invention

Bioreactors can be used as part of an event as a lighting or decorative device, they can serve as lighting and as an interior or exterior information support.

For example, this device can be used as part of a kit intended for individuals. The kit contains a mini-bioreactor of a cylindrical shape, preferably 10 to 30 cm in height and 2 to 10 cm in diameter. It is supplied with air from the base using an integrated compressor. The high-performance nutrient medium is supplied in powder form. The bioluminescent bacteria are supplied in dehydrated form (freeze-dried powder) in a quantity corresponding to 1 ml of culture concentrated at 10-25 DO/ml. The bacteria are cultured by taking the freeze-dried bacteria in the medium prepared by the individual from the powdered medium and tap water.

Claims (7)

Nutrient medium for bioluminescent bacteria comprising:

1. At least one nitrogen source selected from a yeast extract or yeast peptone, the total concentration of nitrogen source being at least 1 g/L (0.1%), preferably between 1 and 30 g/L (0.1 – 3%);
2. At least one carbon source selected from (i) glycerol or (ii) sodium gluconate, the total concentration of carbon source being at least 3 g/L (0.3%), preferably between 3 and 10 g/L (0.3 – 1%) for glycerol and between 6 and 12 g/L (0.6 – 1.2%) for sodium gluconate;
3. A salt selected from (i) NaCl at a concentration of at least 20 g/L (2%), preferably between 20 and 40 g/L (2 – 4%), when the carbon source is glycerol or (ii) NaCl at a concentration of at least 20 g/L (2%), preferably between 20 and 40 g/L (2 – 4%), or KCl at a concentration of at least 20 g/L (2%), preferably between 20 and 30 g/L (2 – 3%), or sea salt at a concentration of at least 38 g/L (3.8%), preferably between 38 and 40 g/L (3.8 – 4%), when the carbon source is sodium gluconate.

A nutrient medium according to claim 1 wherein (i) said salt is sodium chloride and said carbon source is sodium gluconate, or (ii) said salt is potassium chloride and said carbon source is sodium gluconate, or (iii) said salt is sea salt and said carbon source is sodium gluconate or (iv) said salt is sodium chloride and said carbon source is glycerol. Nutrient medium according to one of claims 1 or 2 in powder form. Use of a nutrient medium as defined in one of claims 1 to 3 for cultivating photobacteria of the species Photobacterium kishitanii or Photobacterium phosphoreum . Luminous device comprising a container with a height of at least ten centimeters comprising at least one transparent wall and having an oxygenation system, a nutrient medium according to one of claims 1 to 2 and at least one bioluminescent bacterium chosen from a bacterium of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum . Kit for the production of bioluminescence comprising a container with a height of at least ten centimeters comprising an oxygenation system, a nutrient medium in powder form as defined in claim 3 and bioluminescent bacteria of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum in freeze-dried form. Method for implementing bioluminescence comprising the following steps:

• Place in a container comprising a city water oxygenation system and a nutrient medium as defined in claim 3 in order to reconstitute a liquid nutrient medium;
• Add bioluminescent bacteria of the species Photobacterium kishitanii or of the species Photobacterium phosphoreum in freeze-dried form;
• Activate said oxygenation system;
• Cultivate the bacteria for at least 10 hours to see the light appear;
• Continue culture for desired duration or until bioluminescence extends.