BIO-CONTROL TO FIGHT AGAINST BIO-CONTAMINATION OF FUEL TANKS

Abstract

BIOCONTROL TO COMBAT BIOCONTAMINATION OF FUEL TANKS The present invention relates to the use of microorganisms as biocontrol agents for the prevention and / or treatment of biocontamination in a fuel tank. The present invention particularly relates to a method for preventing and / or treating biocontamination in a fuel tank, comprising introducing at least one biocontrol agent into the tank. The present invention also relates to a method for selecting at least one biocontrol agent, as well as a composition and a kit comprising a biocontrol agent. Figure for abstract: none

Description

Title of the invention: BIO-CONTROL TO COMBAT BIO-CONTAMINATION OF FUEL TANKS Field of invention

[0001] The present invention relates to the treatment of bio-contaminations in fuel tanks. Technological background

[0002] Bio-contamination of fuel tanks is a common phenomenon that affects many sectors of activity (storage of fuel before distribution or use, or storage in machines such as boats, agricultural machines, aircraft, road machines, etc.). It is indeed common for an aqueous phase to appear when combustible petroleum products are stored. Conditions favorable to the development of microbial contamination are thus created: fuels, rich in carbon, provide an essential source of nutrients for the bacteria, fungi and yeasts that live in the aqueous phase. Whatever the form of fuel storage, the multiplication of these microorganisms is always detrimental, due to the resulting risks of bio-corrosion, filter clogging, orifice or gauge obstruction, etc.

[0003] To combat microbial developments and the problematic consequences they can cause, several strategies have been considered, such as the development of biocidal coatings, the addition of biocides, the use of superhydrophobic surfaces promoting water evacuation. However, these different strategies have shown limitations in terms of effectiveness or implementation or cannot be used due to the ban on certain biocides, in particular by the European BPR (Biocide Product Regulation) regulation.

[0004] To date, the only truly effective solution for the treatment of a bio contamination is the mechanical cleaning of the tanks. However, this solution is very restrictive and does not prevent the appearance of new biocontaminations.

[0005] There is therefore a real need to provide solutions that can both treat and prevent bio-contaminations in fuel tanks, these solutions preferably being easy to implement. Description of the invention

[0006] Surprisingly, the inventors have demonstrated that it is possible to prevent and / or treat bio-contamination in a fuel tank. using microorganisms as biocontrol agents. These "good" microorganisms, subsequently called "biocontrol agents", inhibit the growth of "bad" microorganisms responsible for damage to the tank and the fuel itself. These "bad" microorganisms are subsequently called "contaminating microorganisms".

[0007] In order to prevent and / or treat bio-contamination, the bio-control agent(s) are simply introduced into the fuel tank, preferably after prior cleaning of the tank in the event of significant bio-contamination. It is possible to use a single bio-control agent, i.e. a single microorganism, or a mixture of at least two bio-control agents, i.e. at least two microorganisms, in particular of a different species and / or genus. The bio-control agent(s) may be provided in the form of a liquid composition which is simply sprayed inside the fuel tank, before it is filled. Alternatively, the bio-control agent(s) are mixed directly with the fuel before it is introduced.

[0008] The method according to the invention has the particular advantage of being simple to implement.

[0009] A first object of the invention thus relates to a method for selecting at least one biocontrol agent useful for the prevention and / or treatment of biocontamination in a fuel tank, said method comprising: a. provide at least one microorganism to be tested, b. culturing said microorganism to be tested or each of said microorganisms to be tested with a fuel tank contaminating microorganism or with a mixture of at least two fuel tank contaminating microorganisms, and c. selecting, as biocontrol agent, at least one microorganism capable of inhibiting the growth of said contaminating microorganism or microorganisms contaminating said mixture.

[0010] The fuel tank contaminating microorganism used in the selection method may be a fungus, a yeast and a bacterium. Said fuel tank contaminating microorganism is for example: - a fungus of a genus selected from the group consisting of Amorphotheca, Acremonium, Aspergillus, Altemaria, Hypocreales, Discophaerina, Exophiala, Fusarium, Helminthosporium, Paecilomyces, Penicillium, Phialophora, Rhinocladiella, Trichosporium and Trichoderma, - a yeast of a genus selected from the group consisting of Aureobasidium, Candida and Rhodotorula, or - a bacterium of a genus selected from the group consisting of Micrococcus, Staphylococcus, Acinetobacter, Arthrobacter, Aerobacter, Aeromonas, Alcaligenes, Brevibacterium, Desulfovibrio, Dietzia, Escherichia, Enterobacter, Flavobactrium, Kocuria, Leucobacter, Pantoea, Pseudomonas, Streptomyces, Sphingomonas, Serratia and Alicyclobacillus.

[0011] The microorganism to be tested in the selection method which is provided in step a) is preferably a microorganism isolated from an uncontaminated fuel tank and / or expressing at least one fungicidal compound and / or expressing at least one bactericidal compound and / or expressing at least one yeasticidal compound.

[0012] The microorganism selected as biocontrol agent in step c) of the selection method further preferably has at least one of the following properties, preferably at least four of the following properties: - said microorganism is capable of multiplying on a carbon substrate consisting of a fuel, - said microorganism survives in a temperature range from - 40°C to 60°C, preferably from - 40°C to 70°C, more preferably from -40°C to 80°C, - the said microorganism is not pathogenic for humans, - said microorganism is not toxic to fuel, and / or - the said microorganism is not toxic to the reservoir.

[0013] Another subject of the invention relates to a composition comprising at least one biocontrol agent, said biocontrol agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

[0014] Another object of the invention relates to a kit for the prevention and / or treatment of bio-contamination in a fuel tank, said kit comprising: - a first composition comprising at least one biocontrol agent, said biocontrol agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks, and - a second composition comprising at least one formulating agent.

[0015] Another subject of the invention is a method for preventing and / or treating bio-contamination in a fuel tank, said method comprising: a. optionally, clean the fuel tank, and b. introduce into the fuel tank at least one agent of biocontrol, said biocontrol agent being a microorganism capable to inhibit the growth of at least one microorganism contaminating fuel tanks.

[0016] Said biocontrol agent is preferably a microorganism of the genus Bacillus, preferably selected from the group consisting of Bacillus subtilis, Bacillus velenzensis and Bacillus altitudinis.

[0017] Said biocontrol agent is preferably provided in the form of a composition further comprising at least one formulating agent.

[0018] Another object of the invention is the use (i) of at least one bio-control agent, said bio-control agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks or (ii) of a kit as defined above, for the prevention and / or treatment of bio-contamination in a fuel tank.

[0019] Another object of the invention is the use of a microorganism as a bio-control agent for the prevention and / or treatment of bio-contamination in a fuel tank.

[0020] Tank, fuel and contaminating microorganism

[0021] The tank is a container suitable for receiving, storing and / or returning fuel.

[0022] By “fuel” is meant here a fuel comprising hydrocarbons.

[0023] The fuel is for example selected from the group consisting of kerosene, gasoline and diesel.

[0024] The fuel is preferably kerosene.

[0025] The tank can be of any size, depending in particular on its use.

[0026] The reservoir may for example have a volume of at least one liter, for example at at least 10 liters, at least 20 liters or at least 30 liters.

[0027] The tank may have a significantly larger volume, for example a volume of at least 1000 liters, at least 3000 liters, at least 30,000 liters or at least 100,000 liters, for example in the case of an aircraft fuel tank.

[0028] The tank may be made of any material suitable for storing fuel, for example light alloy, such as aluminum alloy or stainless steel and / or mastic, such as polysulfide or polythioether, with for example epoxy or polyurethane paint.

[0029] The tank preferably further comprises at least one element selected from the group consisting of a circuit, an injector, an engine filter, a gauge and a pump.

[0030] The tank may be integrated into or form part of a vehicle, such as a land, sea, air or space vehicle.

[0031] By "contaminated fuel tank" is meant here a tank comprising microorganisms which are toxic to the fuel and / or to the fuel tank.

[0032] A microorganism toxic to a fuel and / or to a fuel tank is hereinafter called a “fuel tank contaminating microorganism” or “contaminating microorganism”.

[0033] A microorganism toxic to a fuel is a microorganism that alters at least one property of a fuel, said property of the fuel being for example selected from the group consisting of its composition (in particular acidity and / or, aromatic compound content), its volatility (in particular distillation temperature, final boiling point, flash point, distillation residue, distillation loss and / or density at 15°C), its fluidity (in particular freezing point and / or viscosity at -20°C), combustion quality (net heat of combustion and / or smoke point), its thermal stability, the absence of contaminants and its electrical conductivity.

[0034] The properties of the fuel can be evaluated by any suitable method well known to those skilled in the art, for example as described in ASTM D1655 and, optionally, in ASTM D4054.

[0035] The composition of the fuel is altered if at least one compound of the fuel is degraded. The degradation of at least one compound of the fuel results, for example, from the production of organic acids and / or the secretion of acidic exopolysaccharides by the contaminating microorganism or from the production of hydrogen sulfide by the contaminating microorganism, when it is a sulfate-reducing microorganism.

[0036] A microorganism toxic to a fuel tank is a microorganism which degrades the tank, for example by generating bio-corrosion and / or by forming a dense biofilm capable of obstructing one or more elements of the tank, such as the circuit, injector, engine filter and / or gauge.

[0037] A contaminated fuel tank is thus characterized in particular by the presence of corrosion inside the tank and / or by the presence of dense biofilms.

[0038] By “corrosion” we mean here the alteration of a material by chemical reaction.

[0039] By "bio-corrosion" we mean here the corrosion of a material caused by micro organisms. Biocorrosion results, for example, from the production of acids by the metabolism of the microorganism that are involved in corrosive chemical reactions. Biocorrosion is accentuated during the formation of a biofilm that concentrates the acids in one place and / or generates differential aerations by the juxtaposition of aerobic and anaerobic microorganisms. Biocorrosion can also result from the degradation of tank polymers (for example, the polymers of the sealant and / or the polymers of the tank paint), thus weakening the protective film of the tank bottoms, walls or metal equipment.

[0040] The term "biofilm" here refers to a community of microorganisms adhering to each other and to a surface, generally to a surface in contact with water. The biofilm comprises microorganisms and an adhesive and protective extracellular matrix. This extracellular matrix is ​​secreted by these microorganisms. The biofilm thus allows the microorganisms to attach to a surface, in particular here to the internal surface of the tank and / or to the surface of the elements of the tank.

[0041] By “dense biofilm” is meant here a biofilm capable of obstructing an orifice.

[0042] Many different microorganisms have been identified in contaminated fuel tanks.

[0043] The microorganism contaminating fuel tanks may be a fungus, yeast or bacteria.

[0044] The microorganism contaminating fuel tanks is for example: - a fungus of a genus selected from the group consisting of Amorphotheca, Acremonium, Aspergillus, Altemaria, Hypocreales, Discophaerina, Exophiala, Fusarium, Helminthosporium, Paecilomyces, Penicillium, Phialophora, Rhinocladiella, Trichosporium and Trichoderma, - a yeast of a genus selected from the group consisting of Aureobasidium, Candida and Rhodotorula, or - a bacterium of a genus selected from the group consisting of Micrococcus, Staphylococcus, Brachybacterium, Aerococcus, Acinetobacter, Arthrobacter, Aerobacter, Aeromonas, Alcaligenes, Brevibacterium, Desulfovibrio, Dietzia, Escherichia, Enterobacter, Flavobactrium, Kocuria, Leucobacter, Pantoea, Pseudomonas, Streptomyces, Sphingomonas, Serratia and Alicyclobacillus.

[0045] The fungus of the genus Amorphotheca is for example Amorphotheca resinae (also called Hormoconis resinae).

[0046] The fungus of the genus Penicillium is preferably selected from the group consisting of Penicillium chrysogenum, Penicillium spindosum and Penicillium corylophilum.

[0047] The fungus of the genus Altemaria is for example Altemaria hordeiaustralica. The mushroom of the genus Acremonium is for example Acremonium strictum.

[0049] The fungus of the genus Aspergillus is for example Aspergillus niger or Aspergillus fumigatus.

[0050] The mushroom of the genus Discophaerina is for example Discophaerina fagi.

[0051] The mushroom of the Exophiala genus is for example Exophiala jeanselmei.

[0052] The fungus of the genus Fusarium is for example Fusarium moniliforme.

[0053] The fungus of the genus Paecilomyces is for example Paecilomyces variotii.

[0054] The fungus of the genus Phialophora is for example Phialophora richardsia.

[0055] The fungus of the genus Trichoderma is for example Trichoderma viride.

[0056] Yeast of the genus Aureobasidium is for example Aureobasidium pullulons.

[0057] Yeast of the genus Candida is for example Candida famata or Candida lipolytica.

[0058] The yeast of the genus Rhodotorula is for example Rhodotorula mucilaginosa.

[0059] The bacteria contaminating reservoirs is preferably a sulfate-reducing bacterium and / or one releasing acid metabolites.

[0060] The bacterium of the genus Micrococcus is for example Micrococcus aloeverae. The bacterium of the Staphylococcus genus is for example Staphylococcus wameri or Staphylococcus vitulinus.

[0062] The bacterium of the genus Acinetobacter is for example Acinetobacter calcoaceticus or Acinetobacter cerificans.

[0063] The bacterium of the genus Arthrobacter is for example Arthrobacter Cummins ii.

[0064] The bacteria of the genus Aerobacter is for example Aerobacter aerogenes. The bacterium of the Brevibacterium genus is for example Brevibacterium ammoniagenes.

[0066] The bacterium of the genus Desulfovibrio is for example Desulfovibrio desulfuricans.

[0067] The bacterium of the genus Dietzia is for example Dietzia cinnamea.

[0068] The bacterium of the genus Enterobacter is for example Enterobacter cloacae or glomerans.

[0069] The bacteria of the genus Flavobacterium are for example Flavobacterium arborescens or Flavobacterium diffusum.

[0070] The bacterium of the genus Kocuria is for example Kocuria rhizophilia.

[0071] The bacterium of the genus Leucobacter is for example Leucobacter komagatae.

[0072] The bacterium of the genus Pantoea is for example Pantoea ananatis.

[0073] The bacterium of the genus Pseudomonas is for example Pseudomonas aeruginosa.

[0074] The bacterium of the genus Streptomyces is for example Streptomyces argenteolus, Streptomyces orientalis or Streptomyces pulcher.

[0075] The bacterium of the genus Sphingomonas is for example Sphingomonas zeae or Sphingomonas aeria.

[0076] The bacterium of the genus Serratia is for example Serratia marcescens or Serratia odorifera.

[0077] The bacterium of the genus Alicyclobacillus is for example Alicyclobacillus acidocaldarius.

[0078] A contaminated fuel tank generally comprises at least one contaminating microorganism selected from the group consisting of Amorphotheca Resinae, Aspergillus Niger, Penicillium chrysogenum, Penicillium spindosum, Altemaria hordeiaustralica, Micrococcus aloeverae and Staphylococcus wameri.

[0079] A contaminated reservoir most often includes the contaminating microorganism Hormoconis resinae.

[0080] An uncontaminated fuel tank also includes microorganisms, but these are not toxic to the tank or to the fuel. In particular, microorganisms present in uncontaminated tanks do not form dense biofilms. An uncontaminated fuel tank is characterized by the absence of dense biofilm, the absence of corrosion and the absence of biodegradation of the tank polymers. Biocontrol agent

[0081] The present invention particularly relates to a bio-control agent, in particular useful for the prevention and / or treatment of bio-contamination in a fuel tank, in particular in a kerosene tank.

[0082] By “biocontrol agent” is meant here a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

[0083] The microorganism contaminating fuel tanks is in particular as defined above.

[0084] Growth inhibition may be total or partial.

[0085] In the case of partial growth inhibition, said growth inhibition is sufficient to prevent the contaminating microorganism from generating corrosion and / or forming a dense biofilm. The partial inhibition may in particular result from a biostatic effect of the biocontrol agent.

[0086] Depending on whether the contaminating microorganism is a bacterium, a yeast or a fungus, the biostatic effect is respectively a bacteriostatic, yeastostatic or fungistatic effect.

[0087] A biostatic effect means a slowing down of the growth of the contaminating microorganism.

[0088] Total growth inhibition may result from a biocidal effect of the biocontrol agent.

[0089] Depending on whether the contaminating microorganism is a bacterium, a yeast or a fungus, the biocidal effect is respectively a bactericidal, yeasticidal or fungicidal effect.

[0090] A biocidal effect means that the contaminating microorganism is killed.

[0091] The biocontrol agent is preferably selected by a selection method as defined below.

[0092] The biocontrol agent preferably has at least one of the following properties, more preferably at least two of the following properties, more preferably still at least four of the following properties: - it is capable of multiplying on a carbon substrate consisting of a fuel, - it survives in a temperature range from -40°C to 60°C, preferably from -40°C to 70°C, more preferably from -40°C to 80°C, - it is not pathogenic for humans, - it is not toxic to fuel, and / or - it is not toxic to the tank.

[0093] Preferably, the biocontrol agent as defined above has at least the following three properties: - it is not pathogenic for humans, - it is not toxic to fuel, and - it is not toxic to the tank.

[0094] The biocontrol agent may be derived from a sample taken from an uncontaminated fuel tank. In this case, the biocontrol agent may have been isolated from said sample and then multiplied. Alternatively, the sample taken from an uncontaminated fuel tank is used directly as a source of biocontrol agents, for example by direct transfer into a contaminated or uncontaminated tank, or the sample taken from an uncontaminated fuel tank is cultured before being used as a source of biocontrol agents.

[0095] The biocontrol agent is preferably a pure strain.

[0096] By “pure strain” we mean a population in which all the individuals are identical.

[0097] The biocontrol agent may be a bacterium, a yeast or a fungus.

[0098] The biocontrol agent is preferably a bacterium.

[0099] The biocontrol agent is preferably a bacterium of the genus Bacillus. The agent biocontrol is preferably a bacterium selected from the group consisting of Bacillus subtilis, Bacillus velenzensis and Bacillus altitudinis.

[0100] The biocontrol agent is for example a strain of Bacillus velenzensis, a strain of Bacillus subtilis or a strain of Bacillus altitudinis, said strain having been isolated from an uncontaminated fuel tank.

[0101] Method for selecting at least one biocontrol agent

[0102] The present invention particularly relates to a method for selecting at least one biocontrol agent useful for the prevention and / or treatment of biocontamination in a fuel tank, in particular in a kerosene tank.

[0103] By "bio-contamination" is meant here a contamination by one or more microorganisms contaminating fuel tanks. The microorganism(s) contaminating fuel tanks are in particular as defined above.

[0104] Bio-contamination of a fuel tank can be diagnosed in different ways: - visually, for example by detecting bio-corrosion and / or the presence of dense biofilms forming brown residues inside the tank; detection can be carried out when the tank is empty or by boroscopy; and / or - by biological analysis, for example by ATPmetry or culture, in particular on a sample from the tank at the fuel / aqueous phase interface.

[0105] ATPmetry analysis gives an overall indication of microbial activity.

[0106] The treatment of bio-contamination in a fuel tank therefore means the treatment of a contaminated fuel tank. The contaminated fuel tank is in particular as defined above.

[0107] Preventing bio-contamination in a fuel tank therefore means preventing bio-contamination in an initially uncontaminated fuel tank.

[0108] The selection method as defined above preferably comprises: a. providing at least one microorganism to be tested, b. culturing said microorganism to be tested or each of said microorganisms to be tested with a fuel tank contaminating microorganism or with a mixture of at least two fuel tank contaminating microorganisms, and c. selecting, as biocontrol agent, at least one microorganism capable of inhibiting the growth of said contaminating microorganism or microorganisms contaminating said mixture. Step a)

[0109] Step a) comprises providing at least one microorganism to be tested.

[0110] The selection method can in fact make it possible to test a single microorganism or several microorganisms.

[0111] The microorganism to be tested can be selected on the basis of the biocidal effect, in particular the bactericidal and / or fungicidal and / or yeasticidal effect, of the compounds it secretes. It is possible to identify such a microorganism from existing databases and publications or by analyzing its genome to determine whether it can express one or more compounds having a bactericidal and / or fungicidal and / or yeasticidal effect.

[0112] The microorganism to be tested is preferably a microorganism expressing at least one fungicidal compound and / or expressing at least one bactericidal compound and / or expressing at least one yeasticidal compound. Said fungicidal and / or bactericidal and / or yeasticidal compound is preferably excreted by the microorganism.

[0113] The microorganism to be tested may also be a microorganism isolated from an uncontaminated fuel tank. The advantage of such a microorganism is that it is already adapted to a fuel-based substrate, as well as to the temperatures to which a fuel tank is exposed in its usual use.

[0114] The microorganism to be tested is, for example, a bacterium of the genus Bacillus, preferably isolated from an uncontaminated fuel tank.

[0115] The microorganism to be tested does not necessarily express a bactericidal, fungicidal or yeasticidal compound. Step b)

[0116] Step b) comprises culturing said test microorganism or each of said test microorganisms with a fuel tank contaminating microorganism or with a mixture of at least two fuel tank contaminating microorganisms.

[0117] Where more than one test microorganism is provided in step a), step b) may comprise culturing a single test microorganism or a mixture of at least two test microorganisms with (i) one contaminating microorganism or (ii) a mixture of at least two contaminating microorganisms.

[0118] Preferably, step b) comprises the culture of a single microorganism to be tested with (i) a contaminating microorganism or (ii) a mixture of at least two contaminating microorganisms. It is then possible after step c), to carry out a new step b) with a mixture of at least two microorganisms selected in step c).

[0119] When at least two microorganisms are tested in a mixture, these microorganisms can inhibit the growth of the same genus and / or species of contaminating microorganism or of different species and / or genera of contaminating microorganism.

[0120] When at least two microorganisms are tested in a mixture, these microorganisms may have been previously selected individually by the selection method according to the invention.

[0121] The microorganism(s) contaminating fuel tanks may have been isolated from a sample taken from a contaminated fuel tank.

[0122] The microorganism(s) contaminating fuel tanks may be in the form of a sample taken from a contaminated fuel tank.

[0123] The microorganism(s) contaminating fuel tanks may be pure strains, for example commercially available.

[0124] The microorganism(s) contaminating fuel tanks are, for example, as defined above.

[0125] The microorganism contaminating fuel tanks is, for example, a fungus, a yeast or a bacterium.

[0126] The fuel tank contaminating microorganism is preferably selected from the group consisting of Amorphotheca resinae, Aspergillus niger, Penicillium chrysogenum, Penicillium spindosum, Alternaria hordeiaustralica, Micrococcus aloeverae, and Staphylococcus wameri.

[0127] The microorganism contaminating fuel tanks is preferably Amorphotheca resinae or Aspergillus niger,

[0128] The mixture of at least two contaminating microorganisms may, for example, be characteristic of the microorganisms present in a contaminated fuel tank or comprise the most abundant and / or most frequently found microorganisms in a contaminated fuel tank.

[0129] The culture can for example be carried out on a solid culture medium, such as on a petri dish, or in a liquid culture medium.

[0130] The culture conditions are those appropriate for cultivating the microorganism(s) to be tested and / or the contaminating microorganism(s). The culture conditions, in particular the culture medium and the temperature, are well known to those skilled in the art.

[0131] For example, the culture medium may be a rich medium, such as LB (Luria-Bertani medium), TSB (Tryptone Soy Broth) or YEPD (Yeast Extract Peptone Dextrose), optionally supplemented with agar-agar, in particular for making culture agars.

[0132] By way of example, the culture is carried out at a temperature of from 18°C ​​to 37°C, for example from 19°C to 30°C, more preferably at room temperature, for example from 19°C to 23°C.

[0133] Alternatively, the cultivation conditions may approximate the conditions present in a fuel tank in its usual use. For example, the cultivation may be carried out in a liquid phase in contact with fuel used as a carbon source and in a temperature range to which the fuel tank is exposed in its usual use.

[0134] Preferably, the same quantities of microorganism to be tested and of contaminating microorganism are inoculated. The quantities can be expressed in CFU (colony forming unit).

[0135] The quantity of microorganisms is for example between 100 CFU and 107 CFU depending on the microorganism(s) to be cultivated.

[0136] For example, a liquid medium culture of each microorganism is carried out overnight (for approximately 18 hours) and the same volume of the culture thus obtained is inoculated for each microorganism.

[0137] When it is a solid culture, the microorganism(s) to be tested and the contaminating microorganism(s) can be inoculated in the same place or in two separate places.

[0138] Cultivation is preferably carried out until compatibility or incompatibility of the cultured microorganisms is observed.

[0139] By "compatibility" is meant here the fact that the different microorganisms coexist in the culture. In this case, the microorganism(s) to be tested have a biostatic effect on the contaminating microorganism(s).

[0140] By "incompatibility" is meant here the fact that one or more microorganisms grow to the detriment of the other microorganism(s). If the microorganism(s) to be tested grow to the detriment of the contaminating microorganism(s), the microorganism(s) to be tested inhibit the growth of the contaminating microorganism(s), which may result in a biocidal effect.

[0141] The culture is for example carried out for at least two days, preferably at least three days, for example at least five days, at least one week, at least two weeks or at least three weeks. The culture is for example carried out for a period of between three days and one month.

[0142] The microorganism(s) to be tested may be inoculated at the same time as the contaminating microorganism(s).

[0143] The microorganism(s) to be tested may be seeded before the contaminating microorganism(s), in particular to test the effect on the prevention of bio-contamination by this or these contaminating microorganism(s).

[0144] The microorganism(s) to be tested may be seeded after the microorganism(s) contaminating the fuel tanks, in particular to test the effect on the treatment of bio-contamination by this or these contaminating microorganism(s).

[0145] Step b) thus makes it possible to evaluate the biocidal effect (in particular fungicidal, bactericidal or yeasticidal) or the biostatic effect of the microorganism(s) to be tested with respect to the contaminating microorganism(s). Step c)

[0146] Step c) comprises selecting, as biocontrol agent, at least one microorganism capable of inhibiting the growth of said contaminating microorganism or microorganisms contaminating said mixture.

[0147] The inhibition of the growth of said contaminating microorganism(s) may be a partial or total inhibition.

[0148] Growth inhibition can be assessed by determining the surface area occupied by each microorganism or by analyzing the formation of biofilms.

[0149] Growth inhibition can be assessed visually, for example, by the naked eye or by any suitable technique, such as microscopy.

[0150] For example, in microcopy, a marker may be used to label cell membranes or DNA. In this case, growth inhibition is assessed by comparing (i) the result of individual culture of each microorganism (to be tested and contaminant) with (ii) the result of co-culture of the microorganism(s) to be tested with the contaminating microorganism(s).

[0151] In the event of total inhibition, the microorganism(s) to be tested are the only ones present at the end of step b).

[0152] In the case of partial inhibition, the surface area occupied by the contaminating microorganism(s) is less than or equal to that occupied by the microorganism(s) to be tested.

[0153] In the case of partial inhibition, the contaminating microorganism(s) preferably occupy less than 40% of the surface area occupied by all the microorganisms present at the end of step b), more preferably less than 30% or less than 20%. In the case of partial inhibition, the contaminating microorganism(s) no longer form a dense biofilm.

[0154] The microorganism selected as biocontrol agent in step c) preferably also has at least one of the following properties, more preferably at least two of the following properties, more preferably still at least four of the following properties: - said microorganism is capable of multiplying on a carbon substrate consisting of a fuel, - said microorganism survives in a temperature range from - 40°C to 60°C, preferably from - 40°C to 70°C, more preferably from -40°C to 80°C, - the said microorganism is not pathogenic for humans, - said microorganism is not toxic to fuel, and / or - the said microorganism is not toxic to the reservoir.

[0155] The microorganism selected as biocontrol agent in step c) preferably has the following three properties: - the said microorganism is not pathogenic for humans, - said microorganism is not toxic to fuel, and - said microorganism is not toxic to the reservoir, and

[0156] optionally has at least one of the following properties: - said microorganism is capable of multiplying on a carbon substrate consisting of a fuel, and / or - said microorganism survives in a temperature range from - 40°C to 60°C, preferably from - 40°C to 70°C, more preferably from -40°C to 80°C.

[0157] The selection method as defined above can thus comprise: - a step of evaluating at least one property of the microorganism(s) to be tested, before step a), or - a subsequent step of evaluating at least one property of the microorganism(s) selected as biocontrol agent in step c),

[0158] said property being selected from the group consisting of the absence of pathogenicity for humans, the absence of toxicity for the fuel, the absence of toxicity for the tank, the capacity to multiply on a carbon substrate consisting of a fuel and the capacity to survive in a temperature range of - 40°C to 60°C, preferably of - 40°C to 70°C, more preferably of - 40°C to 80°C.

[0159] The ability of a microorganism to multiply on a carbon substrate consisting of a fuel can be evaluated by any suitable method, such as culturing the microorganism in a container comprising fuel and an aqueous phase. For this purpose, for example 105 CFU of the microorganism are introduced into a container comprising water; the fuel is then added and the whole is incubated for at least 2 days, preferably 7 days, for example at a temperature suitable for said microorganism or at more extreme temperatures chosen between -40°C and 80°C. The microorganism is capable of multiplying on the fuel if its population does not decrease.

[0160] The temperature range from -40°C to 80°C corresponds in particular to the range of extreme temperatures to which a fuel tank in an aircraft may be exposed.

[0161] The ability of the microorganism to survive in a temperature range of -40°C to 60°C, preferably -40°C to 70°C, more preferably -40°C to 80°C can be evaluated by any suitable method. For example, the microorganism is exposed to heat stress at -40°C, for example for at least 2 days, then is cultured at room temperature, for example for at least 2 days, then is exposed to heat stress (for example at a temperature of 60°C to 80°C), for example for at least 2 days, and again cultured at room temperature, for example for at least 2 days. The quantity of live microorganisms is then evaluated, for example by spreading dilutions on rich culture medium. Said microorganism survives in a temperature range between -40°C and 60°C, preferably between -40°C and 70°C, more preferably between -40°C and 80°C if it is still isolatable after exposure to the extreme temperatures of the range considered. For extreme temperatures, such as 70°C-80°C, the microorganism generally survives in spore-forming form. The culture medium may be an aqueous phase in contact with a biofuel as a carbon source or a culture medium suitable for the microorganism.

[0162] The microorganism is not pathogenic for humans, if it does not present a risk to human health, in particular when it is handled by humans (including in particular the isolation, culture and packaging of the microorganism and its use in fuel tanks).

[0163] The toxicity of a microorganism to fuel may be assessed by any suitable method. For example, the microorganism is cultured in the aqueous phase of a tank or bioreactor comprising fuel, for example for at least two weeks. If no fuel compound is degraded or if the other properties of the fuel, in particular as defined above, are not altered, then the microorganism is not toxic to the fuel.

[0164] The toxicity of a microorganism to the tank may be assessed by any suitable method. For example, the microorganism is cultured in the aqueous phase of a tank or bioreactor comprising fuel, for example for at least six months. If no corrosion or dense biofilms are observed in the tank or bioreactor, then the microorganism is not toxic to the tank.

[0165] The toxicities of a microorganism to the fuel and to the tank can be tested at the same time.

[0166] If the selected microorganism is not capable of multiplying on a carbon substrate consisting of a fuel and / or of surviving in a temperature range of - 40°C to 60°C, preferably - 40°C to 70°C, more preferably - 40°C to 80°C, or if it is desired to improve the capacities of the selected microorganism, it is possible to carry out one or more steps of adaptation of the microorganism.

[0167] The method as defined above can thus comprise at least one subsequent step of adapting the microorganism selected as biocontrol agent in step c).

[0168] The adaptation step may include culturing the selected microorganism: - in the presence of fuel as the sole carbon source, and / or - at a temperature of - 40°C to 60°C, preferably - 40°C to 70°C, more preferably from -40°C to 80°C, for example (i) between -40°C and 0°C (in particular at -40°C), (ii) between 0°C and 40°C (in particular at 0°C and / or at 20°C) and / or between (i) 40°C and 80°C (in particular at 50°C, 60°C, 70°C and / or 80°C).

[0169] The duration of the adaptation step is for example at least one day, preferably at least 4 days, preferably at least one week.

[0170] The duration of the adaptation step is for example at least one day, preferably at least 4 days, preferably at least one week, per culture condition, in particular for each culture temperature used.

[0171] The adaptation step allows the microorganism to evolve by selecting descendants capable of multiplying under the temperature and / or substrate conditions used.

[0172] The method as defined above may further comprise a step of using the microorganism(s) selected in step c) or obtained after at least one adaptation step as defined above, as a biocontrol agent, in a fuel tank. This step of use comprises, for example, the introduction, inside the fuel tank, of said microorganism(s) selected in step c) or obtained after at least one adaptation step. The introduction of said microorganism(s) inside the tank is in particular as described below in the section “Method for preventing and / or treating biocontamination in a fuel tank”.

[0173] Composition comprising at least one biocontrol agent

[0174] The present invention also relates to a composition comprising at least one biocontrol agent as defined above.

[0175] The biocontrol agent is preferably obtained by the selection method as defined above.

[0176] In one embodiment, the composition as defined above comprises at least two biocontrol agents as defined above, for example two, three, four or at least five biocontrol agents as defined above.

[0177] The composition may be a sample taken from an uncontaminated fuel tank, in particular taken from the aqueous phase / fuel interface.

[0178] The composition can be obtained by culturing a sample taken from an uncontaminated fuel tank, in particular taken from the aqueous phase / fuel interface.

[0179] In one embodiment, the composition as defined above does not comprise a sample taken from an uncontaminated fuel tank, nor a culture of a sample taken from an uncontaminated fuel tank.

[0180] In a preferred embodiment, the biocontrol agent present in the composition makes it possible to inhibit the growth of Hormoconis resinae, Aspergillus niger and / or a sulfate-reducing bacterium or one releasing acid metabolites.

[0181] The biocontrol agent is preferably a bacterium of the genus Bacillus, in particular selected from the group consisting of Bacillus subtilis, Bacillus altitudinis and Bacillus velenzensis.

[0182] The composition may advantageously further comprise at least one formulating agent. The formulating agent is preferably suitable for the survival and / or adhesion of said biocontrol agent(s) in a fuel tank.

[0183] The formulating agent may, for example, promote the adhesion, spraying, dispersion, stability and / or survival of said biocontrol agent(s).

[0184] The formulation agent is for example an adhesion agent.

[0185] The purpose of the adhesion agent is to promote the adhesion of the biocontrol agent(s) to the internal surface of the reservoir and / or to the surface of the element(s) included in the reservoir.

[0186] The adhesion agent is for example a divalent cation (such as Ca2+, Mg2+, Fe2+ or their combinations) or a polymer (such as polylysine(s)).

[0187] The formulating agent may for example be a nutrient.

[0188] The purpose of the nutrient is to enable the biocontrol agent(s) to multiply rapidly, particularly once introduced into a fuel tank. The biocontrol agent(s) will then use the fuel as a carbon substrate for their growth.

[0189] The nutrient is preferably specific for the growth of said biocontrol agent(s). In particular, the nutrient preferably does not promote the growth of one or more contaminating microorganisms. In a preferred embodiment, the nutrient does not promote the growth of Hormoconis resinae and / or Aspergillus niger.

[0190] The formulating agent(s) may be present in the composition in the form of a solution or a powder.

[0191] The composition preferably comprises at least 80%, preferably at least 90%, for example at least 95% of the biocontrol agent or agents, the percentage being a mass percentage, in particular when the composition is in dry form.

[0192] When the composition comprises several biocontrol agents, they are preferably present in equal amounts.

[0193] The composition preferably comprises at least 100 CFU, for example at least 1000 CFU, at least 104 CFU or at least 105 CFU of the or each biocontrol agent.

[0194] The biocontrol agent(s) present in the composition are in active form.

[0195] By “active form” is meant here a biocontrol agent capable of multiplying.

[0196] The biocontrol agent in active form may be in living and / or revivable form.

[0197] The biocontrol agent in revivable form is, for example, in sporulated form.

[0198] Said biocontrol agent(s) are preferably in lyophilized form or in dehydrated spore form.

[0199] The composition as defined above is preferably in powder form, for example in the form of lyophilized powder.

[0200] The present invention also relates to a composition obtained by mixing the composition as defined above, in particular in which the biocontrol agent(s) are in lyophilized form, with water.

[0201] Alternatively, said biocontrol agent(s) are encapsulated in beads or microcapsules or any other suitable support.

[0202] kit for the prevention and / or treatment of bio-contamination in a fuel tank

[0203] The present invention also relates to a kit for the prevention and / or treatment of bio-contamination in a fuel tank, in particular in a kerosene tank, said kit comprising: - a first composition comprising at least one biocontrol agent, said biocontrol agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks, and - a second composition comprising at least one formulating agent, in particular suitable for the survival and / or adhesion of said biocontrol agent(s) of said first composition in a fuel tank.

[0204] The first composition and the formulating agent of the second composition are in particular as defined above in the section “Composition comprising at least one biocontrol agent”.

[0205] The second composition may be in the form of a solution or a powder.

[0206] Method for preventing and / or treating bio-contamination in a fuel tank

[0207] The present invention also relates to a method for preventing and / or treating bio-contamination in a fuel tank, said method comprising: a. optionally, clean the fuel tank, and b. introducing into the tank at least one biocontrol agent, said biocontrol agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

[0208] The tank and the fuel are in particular as defined above.

[0209] The fuel is preferably kerosene.

[0210] The tank is preferably a kerosene tank of an aircraft. Step a)

[0211] Step a) is optional and includes cleaning the fuel tank.

[0212] Step a) is preferably carried out when the fuel tank is contaminated. This step then makes it possible to improve the efficiency of the process.

[0213] Step a) is preferably carried out when the fuel tank has been treated with a biocidal agent.

[0214] Step a) is carried out in particular when the process concerns the treatment of bio-contamination and the prevention of subsequent contamination.

[0215] Step a) may be preceded by a step comprising emptying the tank and / or rinsing the tank, if applicable.

[0216] Cleaning the fuel tank includes in particular cleaning the internal surface of the tank as well as the element(s) present inside the tank.

[0217] The cleaning may be carried out by any suitable means well known to those skilled in the art. The cleaning is preferably mechanical cleaning, such as high pressure cleaning, for example at a pressure of 10 to 30 bars. Step b)

[0218] Step b) comprises introducing into the fuel tank at least one biocontrol agent as defined above. In particular, said biocontrol agent is a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

[0219] In a preferred embodiment, the biocontrol agent makes it possible to inhibit the growth of Hormoconis resinae, Aspergillus niger and / or a sulfate-reducing bacterium or one releasing acid metabolites.

[0220] The biocontrol agent is preferably a bacterium of the genus Bacillus, in particular selected from the group consisting of Bacillus subtilis, Bacillus altitudinis and Bacillus velenzensis.

[0221] The biocontrol agent is preferably obtained by the selection method as defined above.

[0222] Alternatively, the biocontrol agent may also be derived from a sample taken from an uncontaminated fuel tank. In this case, the biocontrol agent biocontrol may have been isolated from the sample and then multiplied, or the sample taken is used directly or after culture.

[0223] The biocontrol agent(s) are for example provided in a composition, in particular as defined above.

[0224] The composition may advantageously further comprise at least one formulating agent. Said formulating agent is in particular as defined above.

[0225] The composition may be a sample taken from an uncontaminated fuel tank, in particular taken from the aqueous phase / fuel interface.

[0226] The composition can be obtained by culturing a sample taken from an uncontaminated fuel tank, in particular taken from the aqueous phase / fuel interface.

[0227] The composition is preferably in liquid form.

[0228] If the composition comprising the biocontrol agent(s) is in solid form, for example in powder form, step b) preferably comprises mixing said composition with a diluent (for example water), in order to obtain a liquid composition.

[0229] The introduction of at least one biocontrol agent inside the fuel tank may be carried out by applying said biocontrol agent(s) to the entire internal surface of the tank and, optionally, to the element(s) included in the tank. In this case, the tank does not include fuel in step b). The composition is preferably applied to the internal surface of the tank and to the element(s) included in the tank by means of a sprayer.

[0230] Alternatively, the introduction of at least one biocontrol agent inside the fuel tank can be carried out by injecting said agent(s) into the tank, in particular at the fuel / air interfaces and / or the fuel / aqueous phase interfaces and / or at the internal surfaces of the tank not immersed in fuel.

[0231] Alternatively, the introduction of at least one biocontrol agent into the fuel tank may be carried out by pre-mixing the biocontrol agent(s) with the fuel and introducing the resulting mixture into the fuel tank. Step c)#

[0232] The method as defined above may comprise a subsequent step c) comprising filling the tank with a fuel, in particular when the tank is empty or incompletely filled in step b).

[0233] Step c) is preferably carried out at least 1 hour, for example at least two hours, at least three hours, at least four hours or at least 5 hours, after step b). Step d)

[0234] The method may comprise a subsequent step d) of checking the absence of biocontamination in the reservoir.

[0235] Bio-contamination of a fuel tank can be diagnosed as indicated above, for example by visual analysis or biological analysis.

[0236] If bio-contamination is detected, step b) or steps a) and b) of the above method may be repeated.

[0237] The step of checking the absence of bio-contamination in the tank can be carried out several times over time.

[0238] The method according to the invention is preferably implemented before each filling of the tank, in particular for prevention purposes, or as soon as biocontamination is diagnosed.

[0239] Preferably, the method for preventing and / or treating bio-contamination in a fuel tank as defined above does not comprise a treatment step with a biocidal or biostatic agent other than a bio-control agent according to the invention.

[0240] Use of a biocontrol agent or a biocontrol agent

[0241] The present invention also relates to the use of (i) at least one biocontrol agent as defined above or (ii) a kit as defined above, for the prevention and / or treatment of biocontamination in a fuel tank, in particular in a kerosene tank.

[0242] All terms are notably as defined above.

[0243] The present invention also relates to the use of at least one microorganism as a biocontrol agent for the prevention and / or treatment of biocontamination in a fuel tank, in particular in a kerosene tank. The present invention relates, for example, to the use of a microorganism as a biocontrol agent or at least two microorganisms as biocontrol agents, for the prevention and / or treatment of biocontamination in a fuel tank. The said microorganism(s) are, for example, selected by the selection method as defined above. The said microorganism(s) used as biocontrol agents are, in particular, capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

[0244] The invention will appear more clearly on reading the description which follows, given solely by way of non-limiting example. Figures

[0245] [Fig-1] [Fig. 1] shows the results of the culture of strain A (Bacillus alliludinis), the culture of strain B (a strain of Hormoconis resinae), the culture of strain C (another strain of Hormoconis resinae), the co-culture of strains A and B and the co-culture of strains A and C, on agar-agar petri dishes, after 3 days, 5 days, 7 days, 2 weeks and one month. For co-cultures, the strains are inoculated at the same location.

[0246] [Fig.2] [Fig.2] shows the confocal microscopy results of the biofilm culture of strain A (Bacillus altitudinis), the culture of strain B (a strain of Hormoconis resinae), the culture of strain C (another strain of Hormoconis resinae), the co-culture of strains A and B and the co-culture of strains A and C. For each condition, on the left is represented a 3D view in which the microorganisms are stained with both Syto9 and WGA, in the center a capture in transmitted light, to observe the boundary between the oil and the medium used and on the right a 2D image showing the submerged biofilm formed by the different microorganisms.

[0247] [Fig.3] [Fig.3] shows the results of one-week (A) or two-week (B) cultures of different Bacillus strains and a Hormoconis resinae strain. "XX": culture of strain X inoculated at two neighboring locations (1st row) or at the same location (2nd row). "XY": co-culture of strains X and Y inoculated at two neighboring locations. "X+Y": co-culture of strains X and Y inoculated at the same location. A: H. resinae strain. B: Bacillus altitudinis strain. C: Bacillus sp. strain. D: Bacillus velezensis strain. E: Bacillus subtilis strain. Examples Materials and methods

[0248] (i) Isolation / Culture of strains

[0249] Samples from aircraft kerosene tanks were cultured on different rich media such as LB (Luria-Bertani), TSB (Tryptone Soy Broth), and YEPD (Yeast Extract Peptone Dextrose). Cultivation was carried out either in liquid medium or on agar media (enriching the rich medium with 1.5% agar). The inoculated agar media were cultured at room temperature (approximately 20°C) for one week to ensure that all microorganisms present in the sample had time to grow. The strains were then isolated for purification on new agar media. The strains were then stored at -80°C after being cultured only in liquid medium and then adjusted with 20% glycerol.

[0250] (ii) Composite medium

[0251] The composite medium is Tryptic Soy Broth (TS; Liofilchem, REF 610053), supplemented with 1% glucose (glu; Fisher, A16828.0E) and 20% yeast extract peptone dextrose in the final volume (YEPD; Yeast Extract Peptone Dextrose Bacto™: ThermoFisher, REF 212750, Peptone Bacto™: ThermoFisher, REF 211677, Glucose). Concentrations and references are detailed in Table 1 below.

[0252] [Tables 1] Medium Concentration (g / L) Reference Tryptic Soy Broth 30 Liofilchem, REF 610053 Glucose 14 Fisher, A16828.0E Bacto™ Yeast extract 2 ThermoFisher, REF 212750 Bacto™ Peptone 4 ThermoFisher, REF 211677 Difco™ Agar 15 ThermoFisher, REF 214530

[0253] Table 1: Composition of the composite medium

[0254] (iii) Storage of microorganisms in beads

[0255] Microorganisms, including bacteria, fungi and yeasts, obtained from an aircraft kerosene tank by Petri dish culture, were carefully preserved for future analyses. The isolated strains were stored at -80°C in commercially available beads. For this purpose, one colony of each microorganism was cultured in 5 mL of composite medium (TS + 1% glu + YEPD at 20% final concentration) in a 50 mL Falcon tube at room temperature with continuous shaking (150 rpm). The incubation period ranged from 2 to 5 days, depending on the strain and the time required for the microorganisms to reach the stationary phase or be well developed.Following incubation, the cultures underwent vigorous shaking, and for some fungal strains, grinding (using the IKA Ultra Turrax t25 after a sterilization process) was necessary to facilitate disaggregation of the organisms for subsequent pipetting. Following the incubation period, 1.5 mL to 3 mL of the cultured mixture (depending on the strain) was collected in an Eppendorf tube. The samples underwent centrifugation at maximum speed for 2 minutes, after which the supernatant was removed, and the pellet was suspended in the preservation solution present in commercial beads (catalog number TS / 80-MX). The pellet was thoroughly mixed with the preservation solution and then cultured with the beads after 15 seconds of shaking. The cultures were then left in the dark for 2 hours to allow adhesion to the beads. After this incubation period, the excess liquid was removed. was removed from the tubes, and the beads were stored at -80°C for later use. This comprehensive protocol ensures effective preservation of microorganisms.

[0256] (iv) Night culture

[0257] In a 12-well plate, a bead recovered from storage at -80°C is cultured in 3 mL of composite medium. The culture is incubated at room temperature on a shaker set at 150 rpm for approximately 20 hours.

[0258] (v) Culture on petri dish

[0259] The strains to be tested were cultured in liquid medium in their respective media (TS for bacteria, YPD for yeasts / fungi) at room temperature until reaching their exponential growth phase. 5 μL of each culture were then deposited either at the same location or 1 cm apart on a mixed agar medium dish (50% TS / 50% YPD). After 3 days to 1 month of culture at room temperature, (1) the phenotype of the macrocolony that developed from an inoculum composed of two microbial types is compared to that of the macrocolonies obtained for each pure strain and (2) the size and phenotype of each of the macrocolonies that developed 1 cm apart from each other are evaluated.

[0260] (vi) Characterization of the Biofilm

[0261] - Experimental Conditions of the Colony

[0262] In a 6-well plate, a 5 mL preparation of TS + 1% glucose + YEPD (20% final concentration), supplemented with 1.5% agar, was made and allowed to dry for one hour under the hood one day before inoculation. For each strain (and in the case of co-culture, a 1:1 ratio was mixed), 5 pL of the overnight culture was inoculated into the middle of the wells of the 1.5% agar plates containing the composite medium. These plates were then incubated at room temperature (approximately 20°C), and images were captured every few days. The interpretation of a mature biofilm was considered after one week of culture.

[0263] To assess the interaction between microorganisms, inoculation was performed so that the inoculation centers between the two points were separated by 1 cm, with a border of 1-2 mm between the two points. This experimental design allowed the exploration and observation of microbial interactions throughout the incubation period.

[0264] - Development of Submerged Biofilm and Confocal Microscopy Assays

[0265] Submerged biofilms were cultured on the surface of 96-well polystyrene microtiter plates with pclear base (Greiner Bio-one, France), allowing high-resolution fluorescence imaging. Each well received 100 pL of TS + 1% glucose + YEPD (20% final concentration), inoculated with 5 pL of a culture overnight, and supplemented with 50 μL of paraffin oil (Sigma-Aldrich, REF: 18512). The microtiter plate was then incubated at room temperature for 3 days.

[0266] For confocal observation, a combination of diluted dyes was applied. Specifically, 25 μL of the dye mixture, containing 2 μL of Syto9 and 100 μL of WGA, prepared in 1 mL of sterile water, was added to each well. Syto9 serves as a green fluorescent nucleic acid dye for live cell staining, highlighting nucleic acids such as DNA and RNA inside cells, proving useful in biofilm studies to visualize microbial cells. In contrast, WGA, derived from wheat germ, is a lectin that specifically binds to N-acetylglucosamine and sialic acid residues on the cell surface. In biofilm studies, WGA serves as a fluorescent dye to visualize extracellular polymeric substances (EPS), critical components for biofilm formation and structural integrity.This approach facilitated efficient staining and visualization of biofilm structures prior to confocal imaging.

[0267] The submerged biofilms were observed using an inverted Leica SP8 AOBS confocal laser scanning microscope (CLSM, LEICA Microsystems, Wetzlar, Germany) at the INRAE ​​MIMA2 platform. The biofilms were scanned with a lOx objective. SYTO 9 excitation was performed at 488 nm with an argon laser, and the emitted fluorescence was recorded in the range of 500 to 600 nm on hybrid detectors. The excitation Results

[0268] (i) Effect of a strain of Bacillus altitudinis isolated from a non-fuel tank contaminated with strains of Hormoconis resinae

[0269] When strain A of Bacillus altitudinis (isolated from a sample in an uncontaminated fuel tank) is associated with a strain of the fungus Hormoconis resinae, only the phenotype of the bacterium is expressed at 2 weeks and 1 month post-inoculation. The bacterium therefore has fungicidal properties against the fungus Hormoconis resinae (see [Fig.l]).

[0270] The same conclusion can be drawn with another observation technique: confocal microscopy. This technique allows the morphology of the biofilms of the different strains to be observed individually: - strain A of the bacterium Bacillus altitudinis has a thin biofilm that forms alveoli, - strain B of the fungus Hormoconis resinae forms a thick, low-density biofilm, and - strain C of the fungus Hormoconis Resinae forms a thin, but dense biofilm.

[0271] When strain A of the bacterium Bacillus altitudinis is associated with one of the strains of Hormoconis resinae, the biofilm obtained has characteristics close to that of the bacterium alone (see [Fig.2]). These results show that in biofilm, the bacterium Bacillus altitudinis inhibits the growth of biofilms of the strains of Hormoconis resinae.

[0272] (ii) Effect of different strains of the genus Bacillus on different strains of bacteria isolated from contaminated kerosene tanks

[0273] Further tests were carried out by incubating the strains at two neighbouring but separate locations on agar medium. The following bacteria were tested:

[0274] - strain D: strain of Bacillus subtilis NDmed from a culture collection,

[0275] - strain A: strain of Bacillus altitudinis from an airplane sample,

[0276] - strain E: strain of Bacillus velezensis from an airplane sample, and

[0277] - strain F: strain of Bacillus velezensis QST713 marketed by Truffaut.

[0278] Each of the above bacteria is therefore inoculated onto petri dishes near various fungi and bacteria isolated from samples taken from contaminated kerosene tanks, namely: - B and C: strains of Hormoconis resinae, - G: Penicillium chrysogenum, - H: Penicillium spindosum, - I: Hypocreales sp., - J: Micrococcus aloeverae, - K: Staphylococcus wameri, and - L: Altemaria hordeiaustralalica.

[0279] As shown in Table 2 below, the bacteria tested show a more or less notable biocidal effect against microorganisms contaminating fuel tanks. Bacillus velezensis bacteria appear to be the most effective.

[0280] [Tables2] BCGHIJKLD ++ + + + 0 ++ 0 ++ A + 0 0 0 0 ++ 0 - E ++ + + 0 + ++ 0 ++ F ++ ++ ++ 0 ++ 0 0 ++

[0281] Table 2: Characterization of existing interactions between different Bacilli (D, A, E, F) and microorganisms isolated from biocontaminated aircraft tanks (B, C, G, H, I, J, K and L)

[0282] ++: presence almost exclusively of the Bacillus strain, +: Bacillus strain majority, 0: the 2 strains interact, -: majority contaminating strain

[0283] The results differ from previous tests concerning strain A of Bacillus altitudinis: when the Hormoconis resinae fungi are not inoculated in the same place as the bacteria, they have time to develop and strengthen; they are more resistant to the fungicidal action of the bacteria, which therefore only seems local, but sufficient for biocontrol.

[0284] (iii) Effect of different strains of the genus Bacillus on a strain of H. resinae

[0285] Further tests were carried out using an overnight culture of a H. resinae bead (A), as described previously. However, for the Bacillus strains tested (B (Bacillus altitudinis), C (Bacillus sp.), D (Bacillus velezensis), and E (Bacillus subtilis)), an overnight culture of each of these strains was performed from a colony picked from a Petri dish and then cultured in 3 mL of composite medium. Each Bacillus culture was diluted only 100 times, before being incubated at room temperature for 5 hours. 5 μL of the resulting cultures were inoculated onto agar medium for testing.

[0286] For co-cultures, 5 μl of the H. resinae culture was placed in the center of an agar medium. After 5 minutes of incubation for absorption into the agar medium, 5 μl of the Bacillus cultures were inoculated either at a neighboring, but distinct, location or at the same location as H. resinae.

[0287] [Fig.3] shows the results of the cultures obtained after one week (A) and two weeks (B). Each strain of Bacillus tested prevented the growth of H. resinae after one week of culture, whether the inoculation of the two strains was carried out in the same place or in neighboring places. These results were confirmed after 2 weeks of culture: only the Bacillus strains developed.

Claims

Claims

1. Kit for the prevention and / or treatment of bio-contamination in a fuel tank comprising: - a first composition comprising at least one bio-control agent, said bio-control agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks, and - a second composition comprising at least one formulating agent.

2. A method of preventing and / or treating bio-contamination in a fuel tank, said method comprising: a. optionally, cleaning the fuel tank, and b. introducing into the fuel tank at least one bio-control agent, said bio-control agent being a microorganism capable of inhibiting the growth of at least one microorganism contaminating fuel tanks.

3. A method according to claim 2, characterized in that said biocontrol agent is a microorganism of the genus Bacillus, preferably selected from the group consisting of Bacillus subtilis, Bacillus velenzensis and Bacillus altitudinis.

4. Method according to claim 2 or 3, characterized in that said biocontrol agent is provided in the form of a composition further comprising at least one formulating agent.

5. Use of a kit according to claim 1, for the prevention and / or treatment of bio-contamination in a fuel tank.

6. Use of a microorganism as a biocontrol agent for the prevention and / or treatment of biocontamination in a fuel tank.

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