FR3075819A1 - NEW ACETOBACTERIUM BACTERIA STRAINS - Google Patents

Abstract

The present invention relates to a group of new strains belonging to the genus Acetobacterium. The present invention more specifically relates to isolated strains of Acetobacterium sp. Bacteria, having the ability to produce by fermentation at least one compound of industrial interest. The invention also relates to methods for producing at least one compound of industrial interest, in particular an alcohol, such as ethanol, and / or a carboxylic acid and / or a salt thereof, such as acetic acid and / or the acetate using at least one of the new isolated strains of the invention. The invention also relates to the use of at least one of the novel strains of the invention for producing said compound of industrial interest, in particular ethanol and / or acetic acid and / or acetate.

Description

FIELD OF THE INVENTION

The present invention is in the field of microbiology. It relates to new strains isolated from bacteria, as well as their use for producing compounds of industrial interest, in particular alcohols such as ethanol, carboxylic acids or their salts such as acetic acid or one of its salts. . The present invention also relates to methods of producing compounds of industrial interest using these new bacterial strains.

PRIOR ART

The majority of compounds of industrial interest, such as carboxylic acids, salts of carboxylic acids and alcohols are produced by chemical synthesis of raw materials derived from petroleum. However, oil resources are limited and on the verge of depletion, which in particular leads to an increase in their operating costs. In addition, the exploitation of these resources is an important source of pollution and contributes to the undesirable greenhouse effect.

Faced with these drawbacks, industrial processes for the production of compounds of industrial interest using renewable resources have been developed. Thus, organic compounds of industrial interest can be produced by fermentation using microorganisms which use such renewable resources as a raw material, in particular waste. The use of microorganisms capable of metabolizing industrial or agricultural waste in these bioprocesses offers the double advantage of producing compounds of interest while making it possible to eliminate waste that is still difficult to recover and / or recycle.

Carbon monoxide (CO), carbon dioxide (CO2) and dihydrogen (H2, sometimes commonly called hydrogen) represent a significant part of industrial and agricultural waste. Particularly difficult to recycle, these gases are often released directly into the atmosphere, thus contributing to air pollution and the greenhouse effect.

One solution consists in exploiting the capacities of certain microorganisms to transform such industrial or agricultural waste into different compounds of industrial interest, in particular carboxylic acids, salts of such acids, and / or alcohols. Indeed, certain anaerobic bacteria are capable of metabolizing carbon monoxide, carbon dioxide and / or dihydrogen, in particular bacteria of the genus

Acetobacterium of the order Clostridiales. Acetobacterium bacteria are anaerobic acetogenic bacteria, because acetate (of formula CH3COO, or CH3COOH in its acetic acid form) is the main byproduct of their anaerobic metabolism. A. woodîî (DSM1030) was the first species of Acetobacterium identified (Balch et al., 1977). It is also the most described. The genus Acetobacterium also has other representatives, including A. malicum (DSM 4132), A. wieringae (DSM 1911), A. psammoiithicum (SMCC / W751), A. fimetarium (DSM 8238) and A. carbinolicum (DSM 2925). Most of these bacteria were initially described as being homoacetogenic, that is to say as being only capable of producing acetic acid and its conjugate base, acetate, by anaerobic oxidation of dihydrogen and reduction of dioxide carbon, according to the equation:

Reaction (1) Acetic acid is used in the synthesis of vinyl acetate, polyvinyl acetate and cellulose acetate, for example. Cellulose acetate can especially be used in the production of photographic films, varnishes, adhesives, explosives or textile fibers. This compound is also used as a plastic material in various fields. Acetic acid is also used as a solvent in processes for the production of terephthalic acid (TPA), a precursor of polyester PET (Polyterephthalate ethylene).

Rare studies show that some of these strains are also capable of producing a mixture of acetate and ethanol, from a source of organic carbon. Thus, A. woodii, A. wieringae and A carbinolicum can produce a mixture of acetate and ethanol from glucose or fructose, under an atmosphere composed of a binary mixture of carbon dioxide and nitrogen (Buschhorn et al., 1989). A. woodii is also capable of producing a mixture of acetate and ethanol with a mixture of formate and carbon monoxide as the substrate (Bertsch and Müller, 2015). Ethanol is a primary alcohol of formula CH3CH2OH presenting a strong industrial interest. Ethanol is mainly used as a biofuel (bioethanol). This compound is also used by the food industry, or as a solvent in perfumery and pharmaceutical dosage. It is mainly produced by alcoholic fermentation of sugar plants using yeasts. However, this type of process is a major generator of CO2, a harmful gas for the environment.

Also, the production of acetate and ethanol, and preferably ethanol, on an industrial scale from CO, CO2 and / or Lh, by a microorganism such

that Acetobacterium constitutes a very interesting prospect, making it possible at the same time to recover industrial or agricultural waste of CO, CO2 and / or H2.

However, the quantities of ethanol produced by the identified Acetobacterium strains, A. woodii, A. wieringae and A carbinolicum, are relatively low and much lower than the quantity of acetate produced (by a factor of at least 5). In addition, the production of ethanol by these strains requires the presence of an organic carbon source, such as a sugar or a carboxylic acid, thereby increasing production costs and the negative impact on the environment (Buschhorn and al., 1989; Bertsch and Müller, 2015).

There is therefore always a need to identify new microorganisms capable of transforming CO, CO2 and / or ΓΗ2, into different compounds of industrial interest.

The present invention makes it possible to meet this need. The present inventors were able to identify new strains isolated from bacteria of the genus Acetobacterium. The inventors have shown that, surprisingly, these new strains are capable of synthesizing significant amounts of ethanol and acetate, from a mixture of CO, CO2 and hh, or a mixture of CO and hb, especially in the absence of any other carbon source. These new strains constitute the first example of bacteria of the genus Acetobacterium having the capacity to produce ethanol in a quantity greater than acetate and in particular in the absence of organic carbon source.

SUMMARY OF THE INVENTION

In the context of the present invention, the inventors have brought to light, quite surprisingly, new strains isolated from bacteria, belonging to the genus Acetobacterium of the order Clostridiales.

The present invention therefore relates to a group of new strains belonging to the genus Acetobacterium.

The inventors have been able to surprisingly show that these new isolated strains have the capacity to produce significant quantities of at least one compound of industrial interest. These new strains can in particular produce significant quantities of at least one alcohol of industrial interest, such as ethanol, and / or significant quantities of at least one carboxylic acid and / or at least one of acid. carboxylic of industrial interest, such as acetic acid and / or acetate.

The present invention relates more specifically to strains isolated from bacteria Acetobacterium sp. (sp. for species or "species"), having the capacity to produce by fermentation at least one compound of industrial interest. The invention relates more particularly to six strains isolated from bacteria Acetobacterium sp. The invention also relates to methods for producing at least one compound of industrial interest, in particular an alcohol, such as ethanol, and / or a carboxylic acid and / or a salt thereof, such as acetic acid. and / or acetate using at least one of the new isolated strains of the invention. The invention also relates to the use of at least one of the new strains of the invention for producing said compound of industrial interest, in particular ethanol and / or acetic acid and / or acetate.

DESCRIPTION OF THE FIGURES

Figure 1 presents a phylogenetic dendogram of the 26 isolated strains (graphic representation made on NCBI Tree View, Neighbor joining).

Figure 2 presents a phylogenetic dendogram of the 6 selected strains (graphic representation made on NCBI Tree View, Neighbor joining).

Figure 3 shows an example of growth curves (DOôoo) and pH, consumption of syngas and production of metabolites (ethanol and acetate) by the strain P4066.

Figure 4 shows an example of growth curves (DOôoo) and pH, consumption of syngas and production of metabolites (ethanol and acetate) by the strain P4073.

Figure 5 shows the growth curves (DOôoo) and pH, consumption of syngas and production of metabolites (ethanol and acetate) by the strain P4074.

FIG. 6 shows an example of growth curves (DOo) and of pH (A), consumption of syngas (B) and production of metabolites (ethanol and acetate; C) by the strain P4066, in minimum medium, with stirring at 180rpm and at 25 ° C.

Figure 7 shows an example of growth curves (DOôoo) and pH (A), consumption of syngas (B) and production of metabolites (ethanol and acetate; C) by the strain P4073, in minimum medium, with stirring. 180rpm and at 25 ° C.

Figure 8 shows an example of growth curves (DOôoo) and pH (A), consumption of syngas (B) and production of metabolites (ethanol and acetate; C) by the strain P4074, in minimum medium, with stirring. 180rpm and at 25 ° C.

Figure 9 shows an example of growth curves (DOôoo) and pH (A), consumption of syngas (B) and production of metabolites (ethanol and acetate; C) by the strain P4112 (A. wieringae), in medium minimum, without agitation (static) and at 30 ° C. DETAILED DESCRIPTION OF THE INVENTION Definitions

By "microorganism" or "microorganism" is meant a living microscopic organism. Microorganisms include bacteria, microscopic fungi, archaeobacteria, protists, microscopic green algae, plankton animals, planaria, and amoebae.

By "bacteria" is meant a living prokaryotic microorganism, derived from any environmental medium.

By “bacteria of the genus Acetobacterium”, “bacteria of Acetobacterium”, “bacteria of Acetobacterium sp.”, Or “Acetobacterium sp.”, Is meant an anaerobic bacterium belonging to the genus Acetobacterium, within the family of Eubacteriaceae, in the order of Clostridiales and the Clostridia class, positive for Gram staining. Acetic acid (or acetate) is the main byproduct of the anaerobic metabolism of bacteria of the genus Acetobacterium. The genus Acetobacterium includes in particular the species A. malicum (DSM4132), A. wieringae (DSM1911), A. woodii (DSM1030), A. fimetarium (DSM8238) and A. carbinolicum (DSM2925).

By "strain" is meant a bacterium belonging to a defined genus. Within the meaning of the present invention, a strain can denote a bacterium belonging to a species or to a subspecies within said defined genus. Indeed, a defined species can comprise several distinct strains, on the basis of different criteria. Within the meaning of the present invention, a strain can also denote a bacterium belonging to said defined genus and the species of which has not been defined. Thus, within the meaning of the present invention, a strain belonging to the same genus can belong to the same species or to a species different from a distinct strain. Within the meaning of the present invention, two strains are distinct if they differ by at least one molecular characteristic, such as the gene sequence coding for 16S ribosomal RNA (rRNA), the content of guanine (G) and cytosine (C) of their genomic DNA, or the sequence of total genomic DNA (determined by hybridization of the genomic DNA of the strains to be compared).

By "isolated strain" is meant a strain of bacteria which is extracted from its natural environment (that is to say separated from at least one other component with which it is naturally associated).

By “fermentation” is meant a metabolic process carried out by a living microorganism, in particular a bacterium, comprising the partial or total conversion (or degradation) of one or more substrates into at least one metabolite or fermentation product, most often by redox.

Fermentation can be carried out in an aerobic atmosphere (in the presence of oxygen, O2) or in an anaerobic atmosphere (in the absence of oxygen).

We can distinguish several types of fermentation according to the fermentation product (s) (also called metabolite) obtained by the implementation of this metabolic process. We speak for example of alcoholic fermentation when the fermentation products obtained consist of a mixture of at least one alcohol, such as ethanol, and gases (most often carbon dioxide, also called carbon dioxide, or carbon dioxide , chemical formula CO2). We can speak of mixed fermentation when a more complex mixture of fermentation products is obtained. This mixture can for example comprise one or more alcohols, such as ethanol, butanol; one or more carboxylic acids or salts thereof, such as acetic acid or acetate, formic acid or formate, butyric acid; lactic acid or lactate; one or more gases, such as dihydrogen (H2), carbon dioxide (CO2), carbon monoxide (CO) or dioxygen (O2).

By "anaerobic atmosphere", "anaerobic environment", "anaerobic condition", or "anaerobic", we mean an atmosphere, an environment, an environment, a container or an enclosure which contains little or no oxygen (O2).

Preferably, the anaerobic atmosphere, the anaerobic environment, the anaerobic condition, or Panaerobiosis, contains less than 1% of oxygen, in volume percentage. More preferably, the anaerobic atmosphere, the anaerobic medium, the anaerobic condition, or Panaerobiosis, contains less than 0.9% of oxygen, preferably still less than 0.8% of oxygen, preferably still less than 0.7 % of oxygen, preferably still less than 0.6% of oxygen, preferably still less than 0.5% of oxygen, preferably still less than 0.4% of oxygen, more preferably less than 0.3% of dioxygen, preferably even less than 0.2% of oxygen, preferably still less than 0.1% of oxygen, preferably still less than 0.09% of oxygen, preferably still less than 0.05% of oxygen, more preferably less than 0.01% dioxygen, more preferably less than 0.005% dioxygen, more preferably less than 0.001% dioxygen, in volume percentage. Even more preferably, the anaerobic atmosphere, the anaerobic environment, the anaerobic condition, or the anaerobic condition, is devoid of (or contains 0%) dioxygen.

By "culture medium" is meant a medium, or support, or environment, allowing the growth of living microorganisms, here bacteria, that is to say a support comprising the components essential for their growth. The culture medium can be in liquid or solid form. Solid media generally have a composition similar to the corresponding liquid culture medium, supplemented with at least one gelling agent, such as agar or agar.

A culture medium can be a minimum medium or a rich medium. A minimum medium, or defined medium, is a medium comprising the chemical elements strictly necessary for the growth of the bacterial strain or of the mixture of bacterial strains of interest. The chemical elements strictly necessary for bacterial growth (or essential elements) are the compounds that the bacteria of interest are not able to synthesize by themselves. The minimum medium is generally a synthetic medium, the exact qualitative and quantitative composition of which is known. A rich medium is a medium comprising all the chemical elements strictly necessary for the growth of the bacterial strain or of the mixture of bacterial strains of interest as well as additional elements necessary for growth but which these bacteria are capable of synthesizing.

By "growth" or "bacterial growth" is meant the phenomenon of increasing or multiplying the number of bacteria initially present in a given culture.

Bacterial growth can be divided into several phases. In general, the characteristics of the different growth phases can be viewed on a graph representing the number of N cells as a function of time t (typically an lnN = f (t) curve). Bacterial growth can in particular comprise an exponential growth phase and / or a stationary phase.

By "exponential growth phase" is meant the period of time or period during which the number of cells increases logarithmically. During this phase, the cell reproduction speed has reached its maximum and remains constant.

By "stationary phase" is meant the period of time or the period during which the number of cells remains constant. Growth therefore appears to be zero. During this phase, the number of cells that divide is equal to the number of cells that die (especially by cell lysis). In general, this phase occurs after the exponential phase. The number of cells is therefore maximum, and remains constant.

By "culture" is meant a culture medium and at least one cell of at least one microorganism belonging to a defined strain. By "biologically pure culture" is meant a culture of a single strain of microorganism. The presence of any living microorganism other than the particular strain thus cultivated is excluded. Thus, the biologically pure culture within the meaning of the present invention comprises a culture medium and at least one cell of a microorganism belonging to a single defined strain, to the exclusion of any living cell belonging to another strain. In practice, the biologically pure culture can be fixed on or contained in an appropriate support.

By "substrate" is meant a substance used as a starting reagent in a chemical, biochemical or biological reaction, which is modified, degraded or converted during said reaction to form one or more substance (s), called substance (s) ( s) final (s) or final product (s), different from the starting substance. The starting substance and / or the final substance / the final product may be an element or a mixture of elements, a molecule or a mixture of molecules, a compound or a mixture of compounds, or a mixture of elements and / or molecules and / or compounds.

“Carbon source” means an element or a mixture of elements, a molecule or a mixture of molecules, a compound or a mixture of compounds containing carbon atoms and which can be used as an energy source by a microorganism alive, for its metabolism and growth. The carbon source can be used directly by the microorganism to extract the carbon therefrom, or can be converted into one or more intermediate compound (s) from which the microorganism can extract the carbon.

By "organic carbon source" is meant a molecule or a compound comprising at least one carbon atom (C), one oxygen atom (O) and one hydrogen atom (H).

By "syngas", "syngas", "synthesis gas" or "synthesis gas" means a gas mixture comprising at least carbon dioxide (CO2) and / or carbon monoxide (CO), and / or dihydrogen (H2). This gas mixture can also include methane (CHU), water vapor (H20), and impurities. This gas mixture can come from industrial, agricultural, municipal or domestic waste. It may also have been synthesized by pyrolysis. Preferably, the syngas comprises carbon dioxide (C02), carbon monoxide (CO), dihydrogen (H2), a mixture of carbon monoxide (CO) and hydrogen (H2), a mixture of carbon dioxide carbon (C02) and hydrogen (H2), or a mixture of carbon dioxide (C02), carbon monoxide (CO) and hydrogen (H2). Preferably, the syngas contains less than 5% of methane (CHU), of water vapor (H2O) and of impurities, in volume percentage, more preferably still less than 4% of methane (CHU), of vapor of water (H2O) and impurities, more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, more preferably less than 0.8%, more preferably less than 0 , 6%, more preferably less than 0.4%, more preferably less than 0.2%, more preferably less than 0.1%, more preferably less than 0.05%, more preferably less than 0 , 01%, preferably even less than 0.005%, preferably even less than 0.001% of methane (CHU), of water vapor (H2O) and of impurities, in volume percentage. Even more preferably, the syngas contains 0% (is free) of methane (CHU), water vapor (H2O) and impurities. The syngas preferably contains between 25 and 50% of H2, between 35 and 60% of CO and / or between 5 and 30% of CO2, in% by volume. More preferably, the syngas consists of CO, or a mixture of CO2, CO and H2, or a mixture of CO and H2, or also a mixture of CO2 and H2. More preferably, the syngas contains 38% of H2, 48% of CO and 14% of CO2, in% by volume.

By "fermentation product", "final fermentation product", "final product", "compound produced by fermentation", or "metabolite", or "final metabolite", or "fermentation metabolite" means a substance obtained by the implementation of a fermentation. This substance can be an element or a mixture of elements, a molecule or a mixture of molecules, a compound or a mixture of compounds, or a mixture of element (s), molecule (s) and / or compound (s) ).

By "alcohol" is meant a compound in which one of the carbon atoms is linked to a hydroxyl group (-OH). Examples of alcohols are methyl alcohol (or methanol), ethyl alcohol (or ethanol), propyl alcohol (or propanol) and butyl alcohol (or butanol). Ethanol is an alcohol of great industrial interest due to its many applications. By "ethanol" or "ethyl alcohol" means an alcohol of semi-developed formula CH3-CH2-OH.

By "carboxylic acid" is meant a compound comprising a carboxyl group (C (O) OH). These are acids and their conjugated bases are called carboxylate ions (they include a carboxylate group (COO-). In solution in water, the acid form partially dissociates into a carboxylate ion, for the most part carboxylic acids, according to equation:

Reaction (2)

Thus, a carboxylic acid can be present in these two forms in an aqueous solution and, in this case, an equilibrium is established between the acid form and the basic form. Examples of carboxylic acids are methanoic acid (or formic acid), ethanoic acid (or acetic acid), propanoic acid (or propionic acid) and butanoic acid (or butyric acid). Their conjugate bases are respectively methanoate (or formate ion, or formate), ethanoate (or acetate ion, or acetate), propanoate (or propionate ion, or propionate) and butanoate (or butyrate ion, or butyrate).

A carboxylic acid or its conjugate base can also be found in complex with an element which allows it. Examples of such elements include alkali metals, such as potassium (K) and sodium (Na), alkaline earth metals, such as calcium (Ca) and magnesium (Mg), and transition metals, such as copper (Cu) and iron (Fe).

By "carboxylic acid salt" is therefore meant a carboxylic acid or its base conjugated in complex with an element. Examples of the carboxylic acid salts include calcium acetate, potassium acetate, magnesium acetate, sodium acetate, copper acetate and iron acetate.

Thus, within the meaning of the present invention, the terms “carboxylic acid” refer both to the carboxylic acid form, to its basic or conjugated ionic form, and to their salts.

Within the meaning of the present invention, the terms “ethanoic acid”, or “acetic acid”, therefore designate both the carboxylic acid of semi-developed formula CH3-CO-OH, its conjugate base, ethanoate, acetate ion, or acetate, of semi-developed formula CH3COO-, and their salts, such as calcium acetate, potassium acetate, sodium acetate, and copper acetate. Similarly, by "acetate", "acetate ion", or "ethanoate" means the acetate ion of semi-developed formula CH3COO-, as well as its

conjugate acid, ethanoic acid, or acetic acid, of semi-developed formula CH3-CO-OH, and their salts.

By "bioreactor", "fermenter" or "propagator" means an apparatus, a tank or an enclosure which can be opened or closed, hermetic or not, suitable for culture, in which or which live microorganisms are cultivated for the production of biomass, and / or for the production of a fermentation product (or metabolite) or also for the bioconversion of a molecule of interest (or substrate). A bioreactor comprises for example a culture, an atmosphere (aerobic or anaerobic), one or more substrate (s), and optionally one or more fermentation product (s).

By "biomass" is meant the total mass of all the microorganisms present in a given medium, system, environment, or bioreactor.

By "batch mode", "batch feeding mode", "batch mode", "batch feeding mode", "batch fermentation", "batch fermentation", "batch fermentation", or "batch culture" means a fermentation carried out in an apparatus, a tank, a system or an enclosure (for example a bioreactor or a fermenter), open or closed, taking place without addition or recovery (or racking) of culture, substrate, nutrients, biomass, or fermentation product. The culture medium is inoculated with microorganisms (called inoculates) at the start of the process. The evolution of the culture conditions can be followed, for example by measuring the pH, the temperature, the pressure, or the optical density, using suitable devices or systems known to man. of career. Batch mode fermentation does not exclude taking samples (of biomass, culture medium, fermentation product, atmosphere, etc.), in particular for monitoring the culture. The content of the culture device is recovered at the end of fermentation.

By “fed-batch mode”, “fed batch mode”, “fed-batch feeding mode”, “continuous-discontinuous mode”, or “fed-batch mode fermentation”, “fed-batch fermentation”, or "Fed-batch culture" means a fermentation carried out in an apparatus, a tank, a system or an enclosure (for example a bioreactor or a fermenter), open or closed, taking place without recovery or racking , and with a continuous or discontinuous feeding with culture medium or a mixture of nutrients. Fermentation in fed-batch mode generally takes place in several phases. At the start of the process, the culture device is partially filled with the culture medium and inoculated with microorganisms (called inoculates). During a first phase, the microorganism multiplies in the initial volume of culture medium. Growth generally starts faster than in batch mode, since the culture volume can be reduced. The concentration obtained can then be higher than in batch mode. During a second phase, culture medium is added to the culture apparatus, continuously or discontinuously as the case may be (continuous or discontinuous feeding). The volume of the culture in the culture apparatus or the fermenter therefore varies continuously. The evolution of the culture conditions can be followed, for example by measuring the pH, the temperature, the pressure, or the optical density, using suitable devices or systems known to man. of career. The fed-batch culture can in particular be used to optimize the conditions for supplying a nutrient liable to have a growth-retarding effect.

By “continuous mode”, “continuous feeding mode”, “fermentation in continuous mode”, “continuous fermentation”, or “continuous culture”, is meant a fermentation carried out in an apparatus, a tank, a system or an enclosure ( for example a bioreactor or a fermenter), open or closed, taking place with a continuous feeding with culture medium or a mixture of nutrients and a continuous recovery or withdrawal of the culture contained in the culture apparatus. In general, the supply and withdrawal rates are identical, in order to keep the culture volume in the apparatus constant. In this case, such a device makes it possible, unlike batch fermentation, to maintain constant and at a desired value the cell concentration, the growth rate of the biomass, the nutritive equilibria of the medium and / or the production of metabolite (s). or desired fermentation product (s). The fermentation in continuous mode can in particular comprise a first step of inoculation of the culture medium with microorganisms. In this case, the culture apparatus is partially or completely filled with the culture medium at the start of the process, and inoculated with microorganisms. A first phase can then be carried out, during which the microorganism multiplies in the initial volume of culture medium, without feeding and / or without racking. This first phase is then comparable to a fermentation in batch mode or to the first phase of a fermentation in fed-batch mode. It allows in particular to allow time for microorganisms to multiply, before the implementation of continuous feeding and / or continuous racking. Once the cell concentration, the growth rate of the biomass, the nutritive equilibria of the medium and / or the production of metabolite (s) or desired fermentation product (s) reached, continuous feeding and continuous racking starts. The evolution of the culture conditions can be followed, for example by measuring the pH, the temperature, the pressure, or the optical density, using suitable devices or systems known to man. of career. Continuous culture can in particular be used to produce biomass or fermentation products in large quantities continuously.

Isolated strain

The inventors have identified, quite surprisingly, a group of 26 new strains of bacteria belonging to the genus Acetobacterium. These new strains are in particular capable of producing compounds of industrial interest, when they are cultured in the presence of an appropriate substrate. The inventors have shown for six of these new strains that they are capable of producing by fermentation at least one alcohol, in particular ethanol, and / or at least one carboxylic acid or a salt thereof, in particular the acetate.

These six strains have been deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, Paris, France), under the respective registration numbers CNCM-l-5192, CNCM-l-5194, CNCM-l-5195 , CNCM-l-5191, CNCM-l-5193, and CNCM-I-5190.

The present invention therefore relates to an isolated strain of bacteria Acetobacterium sp., Characterized in that it has the capacity of a strain chosen from the strains deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, Paris, France ), under the registration numbers CNCM-l-5192, CNCM-l-5194, CNCM-l-5195, CNCM-l-5191, CNCM-l-5193, and CNCM-l-5190, to be produced by fermentation at minus a compound of industrial interest.

According to a preferred embodiment, the isolated strain has the capacity of the strain CNCM-l-5192 or of the strain CNCM-l-5194, to produce by fermentation at least one compound of industrial interest.

By “compound of industrial interest”, is meant a substance which can be used in particular by industry or agriculture, to manufacture, generate or synthesize a consumer product or a substance which can itself be used in particular by industry or agriculture, to manufacture, generate or synthesize a consumer product. A compound of industrial interest therefore preferably has added value vis-à-vis the raw material and the energy required to obtain it. Said substance can be an element or a mixture of elements, a molecule or a mixture of molecules, a compound or a mixture of compounds, or a mixture of element (s), molecule (s) and / or compound (s) ). A compound of industrial interest is for example a fermentation product or a metabolite, or a mixture of these. Alcohols, carboxylic acids, their conjugated bases or their salts are examples of compounds of industrial interest.

By "ability to produce at least one compound of industrial interest by fermentation" is meant the property of a microorganism in synthesizing a compound of industrial interest, for example from a substrate, during a fermentation process.

The inventors have shown that, surprisingly, the six new strains of the invention are in particular capable of rapid growth, can carry out fermentation, and are capable of producing significant quantities of compounds of industrial interest, in particular of the ethanol and acetate. This property is all the more surprising since only three examples of strains of bacteria of the genus Acetobacterium described to date are capable of synthesizing ethanol (A. woodii, A. wieringae and A carbinolicum).

The strains of the invention are in particular capable of switching from one production of a compound of industrial interest to another. The inventors indeed show that, surprisingly, the strains of the invention are capable of producing mainly acetate or ethanol, or a mixture of the two, depending on the culture or fermentation conditions. To date, such a property has not been described for bacteria of the genus Acetobacterium.

In addition, the inventors have shown that these new strains are capable of consuming CO, ΓΗ2 and / or CO2, depending on the culture conditions.

The inventors have shown in particular that, surprisingly, these new strains are capable of synthesizing significant amounts of ethanol and acetate, from a mixture of CO, CO2 and hh, or a mixture of CO and hh, in the absence of any other carbon source. These new strains are the first example of bacteria of the Acetobacterium genus capable of producing ethanol in the absence of any source of organic carbon.

The inventors have also shown that these new strains are capable of producing ethanol in a quantity greater than acetate. This property is all the more surprising since the amounts of ethanol produced by the strains A. woodii, A. wieringae and A carbinolicum of the prior art are relatively small and much lower than the amount of acetate produced (from a factor 5 minimum).

More generally, the present invention relates to an isolated strain of bacteria Acetobacterium sp., Characterized in that it has all the characteristics of industrial interest of a strain chosen from the strains deposited with the CNCM (National Collection of Cultures of Microorganisms , Institut Pasteur, Paris, France), under the registration numbers CNCM-l-5192, CNCM-l-5194, CNCM-l-5195, CNCM-l-5191, CNCM-l-5193, and CNCM-l- 5190. Preferably the strain isolated from Acetobacterium bacteria has all the characteristics of industrial interest of the strain CNCM-l-5192 or of the strain CNCM-l-5194.

The characteristics of industrial interest of an isolated strain according to the invention include, for example, the capacity to produce at least one compound of industrial interest by fermentation, rapid growth, the capacity to consume CO, CO2 and / or ΓΗ2, the ability to synthesize significant amounts of ethanol and acetate from a mixture of CO, CO2 and Fh, or a mixture of CO and Fh, in the absence of any other source of carbon, and / or the ability to produce acetate or ethanol or a mixture of the two depending on the culture conditions, in particular in the absence of an organic carbon source.

According to one embodiment, the isolated strain is chosen from strains CNCM-l-5192, CNCM-l-5194, CNCM-l-5195, CNCM-l-5191, CNCM-l-5193, and CNCM-l-5190 .

According to a preferred embodiment, the isolated strain is the CNCM-1-5192 strain or the CNCM-1-5194 strain.

The present invention further relates to a biologically pure culture of an isolated strain as defined above. Method of producing a compound of industrial interest

The 26 new strains identified by the Inventors are capable of producing compounds of industrial interest, when they are cultured in the presence of an appropriate substrate, in particular by fermentation.

Thus, the present invention relates to a method for producing a compound of industrial interest by fermentation, comprising the steps consisting in: (i) the cultivation of at least one isolated strain as defined above, in the presence of a substrate suitable, in a culture medium and under conditions allowing the growth of said isolated strain, so as to produce said compound of industrial interest; (il) optionally recovering the culture medium comprising said compound, produced in step (i); and (iii) optionally, the purification of said compound from the culture medium recovered in step (ii).

According to one embodiment, the conditions allowing the growth of said isolated strain are fermentation conditions comprising: - a temperature between 20 and 37 ° C, preferably between 22 and 35 ° C, more preferably between 23 and 33 ° C, more preferably between 24 and 31 ° C, more preferably between 25 and 30 ° C more preferably between 26 and 29 ° C, more preferably between 27 and 28 ° C; and / or - agitation, for example orbital, lateral, vertical, horizontal, and / or upward agitation; and / or - an anaerobic atmosphere.

Preferably, the culture of step (i) is carried out in a bioreactor.

According to one embodiment, the culture of step (i) is carried out in batch mode, for example in a bioreactor. According to another embodiment, the culture of step (i) is carried out in fed-batch mode, for example in a bioreactor. According to another embodiment, the culture of step (i) is carried out in continuous mode, for example in a bioreactor. According to a particular embodiment, the culture of step (i) comprises a combination of fermentation or culture carried out in batch, fed-batch and / or continuous mode.

According to one embodiment, the recovery of the culture medium from step (ii) is carried out in batch mode, for example in a bioreactor. According to another embodiment, the recovery of the culture medium from step (ii) is carried out in fed-batch mode, for example in a bioreactor. According to another embodiment, the recovery of the culture medium from step (ii) is carried out in continuous mode, for example in a bioreactor. According to a particular embodiment, the recovery of the culture medium from step (ii) comprises a combination of recoveries carried out in batch, fed-batch and / or continuous mode.

Preferably, when the culture medium is recovered according to step (ii), the recovery mode used corresponds to the culture mode of step (i). Thus, if the culture of stage (i) is carried out in batch mode, the recovery of the culture medium of stage (ii) is preferably carried out in batch mode. In the same way, if the culture of step (i) is carried out in fed-batch mode, the recovery of the culture medium of step (ii) is preferably carried out in fed-batch mode and if the culture of step (i) is carried out in continuous mode, the recovery of the culture medium from step (ii) is preferably carried out in continuous mode.

According to one embodiment, said suitable substrate is introduced or injected into the culture medium into the culture apparatus (for example a bioreactor), for example using a nozzle, a conduit and / or d 'a pipe. According to one embodiment, the substrate is mixed with culture medium before being introduced into or injected into the culture apparatus. Said suitable substrate may be in the liquid state, in the gaseous state, or may be a mixture of liquids and gases.

According to one embodiment, the method for producing a compound of industrial interest by fermentation comprises a step of inoculating the culture medium with at least one isolated strain as defined above (inoculum), carried out before step i ). Preferably, the inoculation is carried out with a biologically pure culture of an isolated strain as defined above, or a sample or a fraction of such a culture. A person skilled in the art will know how to adapt the culture and / or fermentation conditions described in the examples to implement the cultures according to the modes of fermentation he wishes.

According to one embodiment, said suitable substrate comprises a source of carbon. Preferably, said suitable substrate consists of a carbon source.

According to one embodiment, said carbon source is chosen from a sugar, such as glucose, fructose, dextrose; a carboxylic acid or a salt of carboxylic acid, such as acetate, oxaloacetate, formate, fumarate, malate; an alcohol, such as methanol, ethanol, propanol, butanol, pentanol; a diol, such as propane diol, butane diol; ethylene glycol, tetraethylene glycol; polyethylene glycol (PEG); methoxyethanol; ethoxyethanol; trimethoxybenzoate; trimethocycinnamate; acetoin; glycerol; lactate; pyruvate; methoxyacetate; glycolate; caffeate; ethanolamine; choline; trimethylamine; betaine; glycine; the syngas; and any mixture thereof. Said suitable substrate can also be a complex mixture of compounds, such as yeast extract.

According to one embodiment, said suitable substrate is in gaseous form. According to one embodiment, the pressure inside the bioreactor is between 0.5 and 3 bars, preferably between 0.9 and 2.5 bars, more preferably between 1 and 2.4 bars, more preferably between 1.1 and 2.3 bars, more preferably between 1.2 and 2.2 bars, more preferably between 1.5 and 2.1 bars, more preferably between 1.6 and 2 bars.

Preferably, said carbon source comprises, or consists of, a mixture of carbon dioxide (CO2) and dihydrogen (H2), or in a mixture of carbon monoxide (CO) and optionally dihydrogen (H2), or alternatively a mixture of carbon dioxide, dihydrogen and carbon monoxide.

According to a preferred embodiment, said carbon source is syngas, said syngas preferably comprising carbon dioxide (CO2) and dihydrogen (H2), or carbon monoxide (CO) and optionally dihydrogen (H2), or carbon dioxide, dihydrogen and carbon monoxide.

According to a preferred embodiment, said mixture or said syngas is a mixture of CO2, CO and H2. According to another preferred embodiment, said mixture or said syngas is a mixture of CO and hb. According to another preferred embodiment, the syngas consists of a mixture of CO 2 and H2, or of a mixture of CO and H2.

Said syngas or said mixture preferably contains between 25 and 50% of hh, between 35 and 60% of CO and / or between 5 and 30% of CO2, in% by volume. More preferably, said syngas or said mixture contains between 30 and 40% of H2, between 40 and 50% of CO and / or between 10 and 20% of CO2, in% by volume. In a particularly preferred manner, said syngas or said mixture contains 38% of hh, 48% of CO and 14% of CO2, in% by volume. A person skilled in the art will know how to define the composition of the culture media to implement the cultures according to the modes of fermentation of interest.

Said culture medium is for example a minimum medium. The minimum medium is for example a medium devoid of a carbon source in addition to the substrate as defined above. The minimum medium may alternatively comprise an insignificant amount of a carbon source in addition to the substrate as defined above. A non-significant amount of a carbon source in addition to the substrate as defined above consists, for example, of less than 0.1% (weight / volume) of a carbon source in addition to the substrate as defined above. above, more preferably less than 0.07% (weight / volume), more preferably less than 0.05% (weight / volume), more preferably less than 0.03% (weight / volume), more preferably less than 0.01% (weight / volume) of a carbon source in addition to the substrate as defined above.

Said culture medium can for example be a rich medium. A rich medium contains for example at least 0.2% (weight / volume) of a carbon source in addition to the substrate as defined above, preferably at least 0.4% (weight / volume), of more preferably at least 0.6% (weight / volume), more preferably at least 0.8% (weight / volume), more preferably at least 1% (weight / volume), more preferably at least 1.2 % (weight / volume) of a carbon source in addition to the substrate as defined above.

The inventors have shown that, surprisingly, the strains according to the invention are capable of producing by fermentation at least one alcohol, in particular ethanol, and / or at least one carboxylic acid or a salt thereof, especially acetate.

Thus, according to one embodiment, said compound of industrial interest is an alcohol, a carboxylic acid, a salt of carboxylic acid or any mixture of these.

According to a preferred embodiment, said compound of industrial interest is ethanol, acetic acid and / or acetate, a salt of acetic acid, or a mixture of these. In a particularly preferred embodiment, said compound of industrial interest is ethanol.

According to one embodiment, the method comprises step (iii) of purification of said compound of industrial interest from the culture medium recovered in step (ii). A person skilled in the art will be able to define the conditions and the methods for purifying said compound, in particular according to the nature of said compound, the composition of the culture medium and / or the industrial application or not envisaged for said compound.

Use to produce a compound of industrial interest

The present invention relates to the use of an isolated strain as defined above to produce a compound of industrial interest.

According to one embodiment, said compound of industrial interest is produced by fermentation of an appropriate substrate by means of said isolated strain.

Said compound of industrial interest, said suitable substrate and said fermentation conditions are as described above.

EXAMPLES

PART A: AAATERIALS A.1. Minimum middle: middle M238

Table 1: Composition of the minimum medium M238.

When the syngas is used in the M238 medium, there is no addition of Na2S203. A.2. Rich medium: medium M243

Table 2: Composition of the rich medium M243.

To make the minimum media from rich media, organic carbon sources (eg fructose, ethanol, lactate, yeast extract) are removed and replaced by syngas. A3. supplements

Table 3: Composition of the S20 trace element solution.

The trace element solutions are stored at 4 ° C in the dark.

Table 4: Composition of the vitamin S13 solution.

Vitamin solutions are sterilized by filtration (0.2pm) and stored at 4 ° C away from light. A.4. M1 cryopreservation medium

Table 5: Composition of the M1 cryopreservation medium.

A mixture of a volume of a strain culture in exponential growth phase with a volume of cryopreservation medium M1 is produced and distributed in cryogenic tubes which are then stored at -80 ° C.

A. 5. Gaseous substrate: syngas

Table 6: Composition of syngas in% by volume.

PART B: EXAMPLES (Methods and Results)

Example 1: Identification of new bacterial strains B. 1.1. Enhancements

Enrichments have been made from various ecosystems likely to contain acetogenic microorganisms, in order to isolate new strains capable of developing in minimal medium in the presence of syngas as the only source of carbon and energy.

The enrichments were carried out with a minimum medium, M238 (Table 1), the composition of which varies according to the ecosystem tested, at 25 ° C and 37 ° C. As a result, the salinity (0 to 10g / L NaCl) and the pH (5.5-8.5) were adjusted.

A syngas comprising 38% H2 / 48% CO / 14% CO2, in% by volume (Table 6), was chosen as substrate.

Ecosystems were selected based on their microbial diversity, the presence of relevant metabolites, temperature (mesophilic ecosystems) and their origin. Indeed, the 46 ecosystems tested come from water and sediments (e.g. various ecosystems in Auvergne or the Arcachon basin, in France), faeces (e.g. cow, chicken, rabbit, goat, turtle) and of extremophilic ecosystems (ex: hydro thermal spring).

These 46 ecosystems (codified from MC-1 to MC-46) were enriched either at 25 ° C, or at 37 ° C with stirring (180rpm) at 2 bars of syngas, or 92 enrichment conditions. A micro-GC analysis was carried out 1 to 2 times per week, followed by the renewal of the

gas phase. This makes it possible to "rapidly" detect the appearance of strains capable of using syngas and to continue isolation.

Among these 46 ecosystems, 12 presented interesting results in terms of growth and syngas consumption, at 25 ° C and / or at 37 ° C. Among these, 10 ecosystems showed satisfactory growth and consumption of syngas at 25 ° C and 6 ecosystems at 37 ° C, including 4 ecosystems at 25 ° C and 37 ° C (Table 7 below).

Table 7: Growth and consumption results of syngas from preselected ecosystems.

The ecosystem provides, among other things, sources of carbon and energy whose levels are sufficient to ensure bacterial growth during enrichment. It is for this reason that some crops have shown significant growth without consuming syngas.

B. 1.2. isolation

The enrichments with significant growth and consumption of syngas were replicated several times under the same culture conditions in penicillin vials in order to promote the emergence of the desired strains. Depending on the density, diversity and consumption of syngas, several transplanting cycles were necessary.

A series of 10 successive 1 / 10th dilutions in Hungate tubes on agar medium was made from the last positive subculture in a penicillin bottle in the presence of 2 bars of syngas. The growth of the strains was followed by observation of the colonies with a binocular microscope and by monitoring the consumption of the syngas with micro-GC. Colonies were removed using the anaerobic enclosure to best preserve anoxia. Several successive isolations in agar conditions were sometimes necessary to obtain isolated colonies. A final passage into liquid dilution was then carried out. A collection number was then assigned to each isolate and their storage was carried out at -80 ° C in the presence of a cryoprotector (cryopreservation medium, Table 5).

The 26 strains isolated from MC-7, MC-8 and MC-29 enrichments were collected with a collection number ranging from P4060 to P4085. These strains have the capacity to consume syngas as the only source of carbon and energy.

Example 2: Phylogenetic and functional characterization of the 26 new isolated strains B.2.1. Phylogenetic characterization The genomic DNA of the 26 isolated strains was prepared and the gene encoding the 16S rRNA was sequenced. The alignments of the results of these sequencing with the sequence databases made it possible to show that the sequences of the genes coding for the 16S rRNA for these 26 strains are original, with a maximum of 99.06% of identity detected for the strain la closer (Table 8). This result indicates that these 26 strains are all new.

The results further reveal that these 26 new strains are taxonomically close to species belonging to the genus Acetobacterium.

The phylogenetically closest strains identified by this analysis are the following: A. malicum (DSM4132), A. wieringae (DSM1911), A. psammolithicum (SMCC / W751), A. woodii (DSM1030), A. fimetarium (DSM8238) and A. carbinolicum (DSM2925).

The results show that the closest phylogenetically of these 26 new isolated strains is Acetobacterium malicum.

Table 8 details the taxonomic identification results of the new isolated strains.

Table 8: Taxonomic identification of the 26 strains isolated on the basis of 16S rDNA.

The 5 phylogenetically closest strains still available in the collections were ordered from the DSMZ collection (Table 9; A. psammolithicum (SMCC / W751) is no longer available).

Table 9: Collection of the phylogenetically closest strains.

The species belonging to the genus Acetobacterium are described as being homoacetogenic. They are capable of producing acetate from mixtures of Fh + CO2 and using CO as the only source of carbon and energy. They are also homoacetogenic chemoorganotrophs on some substrates. They use the metabolic pathway of tetrahydrofolate to produce acetate.

The taxonomic positioning of these strains is shown in Figure 1.

These 26 new strains could be divided into 6 distinct clusters, according to their taxonomic positioning:

Cluster 1: P4064; - Cluster 2: P4073 and P4075; - Cluster 3: P4076 and P4078;

Cluster 4: P4084 and P4085; - Cluster 5: P4081, P4083, P4060, P4077, P4082, P4066 and P4080; - Cluster 6: P4062, P4065, P4063, P4071, P4067, P4072, P4070, P4068, P4079, P4074, P4069 and P4061. B.2.2. Functional characterization: Production of ethanol and acetate B.2.2.1. Evaluation of strains under the culture conditions used during the isolations

In order to be able to select the most interesting strains, a physiological characterization was carried out. Clostridium ljunsdahlii (P4006) was used as a control strain. The 26 strains were tested on the medium used during their isolation in the presence of 2 bars of syngas.

These tests (conditions not optimized) were carried out in penicillin bottles under 2 bars of syngas (relative pressure) at 25 ° C with stirring at 180 rpm. The kinetics were followed as described below for 3 weeks:

Growth (Optical density at 600nm (DOôoo)) pH (strips)

Syngas consumption (micro-GC)

Production of relevant metabolites such as ethanol and acetate (GC)

The results are presented in Table 10 below:

Table 10: Balance sheet of growth, metabolite production and the ethanol / acetate ratio under non-optimized culture conditions.

NB: the different values in the table were obtained at different culture times. For example, the optima in ethanol concentration and in ethanol / acetate ratio are not simultaneous. In gray: the 6 selected strains (see section B.2.2.2).

The results show that all of the 26 new strains tested show rapid and satisfactory growth under the culture conditions tested.

In addition, these results show that these 26 new strains are all capable of producing ethanol and acetate in the presence of syngas. In addition, a switch from acetate to ethanol was observed for most strains. The maximum ethanol concentrations obtained were 10.6 to 26.7 mM and 9 to 33.1 mM for acetate. The ethanol / acetate molar ratio was 0.8 to 9.4.

These results were compared with the best results obtained for the control strain P4006 (Clostridium ljungdahlii), under optimized culture conditions. Surprisingly, the growth of this strain as well as the maximum production of ethanol is lower for this control strain than for the 26 new strains, with 6.5 mM maximum of ethanol produced under optimized culture conditions. The maximum acetate production was 23.3 mM and the maximum ethanol / acetate molar ratio was 0.3, under the conditions tested.

Thus, the present study has identified 26 new isolated strains capable of producing significant amounts of ethanol and acetate in the presence of syngas. In particular, the ethanol production levels obtained are significantly higher than the maximum ethanol production level obtained for the control strain, Clostridium ljungdahlii, under the conditions tested. These 26 new strains therefore make it possible to implement processes for the bio-production of ethanol and / or acetate from syngas.

B.2.2.2. Selection of strains with the best potential

Six strains were selected from these 26 strains, based on the results. Within each of the 6 clusters identified, the strain with the best potential based on the following criteria: ethanol / acetate ratio, level of ethanol production, level of growth and observation of a switch from acetate production to ethanol, was selected to carry out additional tests (Example 3 below and following). These strains were deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, Paris, France), under the registration numbers indicated in Table 11 below.

Table 11: Provisional registration number at the CNCM.

Example 3: Phylogenetic and physiological characterizations of the 6 selected strains B.3.1. Phylogenetic characterization B. 3.1.1. Taxonomic positioning

The taxonomic positioning of the 6 new selected strains (section B.2.2. Above) was carried out on the basis of the sequences of the gene coding for the 16S rRNA, under the same conditions as in example B.2.1 above. The result is shown in Figure 2. B.3.1.2. Comparison of G + C contents of genomic DNA

The G + C contents (mol%) of the genomic DNA were determined for 3 of the 6 selected strains (P4066, P4073 and P4074). Table 12 below compares the G + C contents (mol%) between these 3 isolated strains and the closest phylogenetically strains (for these, the Inventors used bibliographic data). The results show significant differences between the G + C contents of DNA

genomics of the known strains and that of the 3 new isolated strains (with the exception of A. wieringae).

Table 12: Comparison of G + C% of strains P4066, P4073 and P4074 with that of strains phylogenetically close.

B. 3.1.3. DNA-DNA hybridization

In order to refine the identification results obtained by 16S rDNA sequencing, the total genomic DNA of 3 of the 6 selected strains was hybridized with the total genomic DNA of the closest strain (Acetobacterium malicum DSM 4132) . The results are presented in Table 13.

Table 13: Percentage of DNA / DNA similarity (The means of the measurements carried out in duplicate are in parentheses).

It is generally considered that two strains whose% of genome / genome hybridization is less than 70% belong to different species (Wayne et al., 1987). Thus, the strain type Acetobacterium malicum DSM 4132 (P4104) does not belong to the same species as the strain P4066, nor to the same species as the strain P4073 or as the strain P4074. These 3 strains are therefore affiliated with at least one new species of the genus Acetobacterium.

The total genomic DNA of 3 of the 6 selected strains was then hybridized with the total genomic DNA of the other phylogenetically closest strains identified in example B.2.1 above (P4104: A. malicum (DSM 4132), P4112: A. wieringae (DSM 1911), P4109: A. psammolithicum (SMCC / W751), P4108: A. woodii (DSM 1030), A. fimetarium (DSM 8238) and P4110: A. carbinolicum (DSM 2925).

Table 14 below summarizes the DNA-DNA hybridization results obtained.

Table 14: DNA-DNA hybridization results

These results show that the strains P4066, P4073 or P4074 do not belong to any of the species identified as being the closest phylogenetically strains. These 3 strains therefore represent a new species of the genus Acetobacterium. B.3.2. Physiological characterization B.3.2.1. Morphology

The morphology of the 6 selected strains was studied by microscopy. Table 15 presents the morphological characteristics observed.

Table 15: Morphology of strains.

B.3.2.2. Syngas substitution with a soluble organic compound

Tests were carried out with strains P4064, P4066, P4073, P4074, P4076 and P4085 in order to replace the syngas with a soluble organic compound: M238 (1 or 20X of FeSC> 4.7H20), pH 7 or 8, at 25 ° C: organic carbon sources did not allow significant growth of these 6 strains to be obtained. M238 at 30 ° C: strains P4064, P4066, P4073 and P4074 did not show significant growth in the presence of fructose.

The results are presented in Table 16.

Table 16: Substitution of syngas with an organic substrate.

These results highlight significant differences in growth between the strains, depending on the substrate used. B.3.2.3. Functional characterization of the 6 selected strains a) Evaluation of the strains under the culture conditions used during the isolations

The growth of the 6 selected strains was evaluated under the conditions described in section B.2.2.1 above.

Figures 3 to 5 show the results obtained for 3 of these strains (P4066, P4073 and P4074).

During the exponential growth phase (0 to 110 h) of the P4066, P4073 and P4074 strains, significant consumption of CO and slightly less H2, as well as production of CO2, were observed.

Acetate production took place during the exponential growth phase. Surprisingly, during this phase, a switch from acetate to ethanol was observed. In fact, at the start of the stationary phase, the acetate concentration decreased and the ethanol concentration increased.

Once the ethanol concentration was stationary, the strains again accumulated acetate. Thus, it is possible to adjust the culture time of these new strains to the type of final product desired (ethanol or acetate). Alternatively, the culture medium can therefore be recovered at different times, depending on the final product of interest.

The ethanol / acetate (molar) ratios reached were 2.8, 9.4 and 1.9 respectively for the new strains P4066, P4073 and P4074. So, surprisingly, these new strains are capable of producing larger amounts of ethanol than acetate, after this switchover.

The switch from acetate production to ethanol production coincided with a decrease in CO consumption and a sharp decrease in CO2 production. The P4073 strain presented the most relevant results in terms of growth and production of ethanol and acetate. B) Small volume culture tests

The culture conditions tested are presented below. The compositions of the culture media are described in Example 1 above.

1st transplant (1R) in 5mL of medium + 1mL of inoculum from a cryotube stored at -80 ° C

ο P4066, P4073 and P4074: M238 (minimum medium) + syngas (2 bars relative pressure) at 25 0 C

ο P4104, P4108 and P4112: M243 (rich medium) at 30 ° C ο P4109: M244 (rich medium) at 30 ° C ο P4110: M245 (rich medium) at 30 ° C 2nd and 3rd transplanting (2R and 3R) in 5 mL of medium (see above) + inoculum in exponential growth phase (10%, v / v) from the previous subculture. From the 4th subculture (4R), three successive subcultures (adaptation of the strain's metabolism) under the culture conditions described below:

All strains: M238 + syngas (2 bars relative pressure)

Only for types P4104, P4108, P4112 and P4109: rich culture media by substituting organic carbon sources with syngas (2 bar relative pressure). Control: typical strains: rich medium to validate the viability of the inoculum.

The 4th and 5th subcultures (4R and 5R) were carried out in Hungate tubes (5mL). The growth results during these 2 subcultures are presented in Table 17.

Table 17: Growth results of the strains with the media tested (rich: organic carbon sources; -: no significant growth; ++: dense culture; nd.: Not determined).

The 4 control strains P4104 (A malicum), P4108 (A woodii), P4109 (A carbinolicum) and P4112 (A wieringae) showed significant growth with rich media. In contrast, strain P4110 (A. fimetarium) did not show sufficiently significant growth in any of the media tested. This strain was therefore not used in the rest of this study.

Under the culture conditions described above, only the control strains P4109 (A. carbonilicum DSM 2925) and P4112 (A. wieringae DSM 1911) were able to use syngas as the only carbon source:

P4109: M244 without organic carbon source + syngas (2 bar relative pressure) and M238 + syngas (2 bar relative pressure) at 30 ° C P4112: M238 + syngas (2 bar relative pressure) at 30 ° C.

The other 2 control strains P4104 (A malicum DSM 4132) and P4108 (A woodii DSM 1030) did not show significant growth in the presence of syngas as the only source of carbon and energy, under the culture conditions tested. The presence of CO (a toxic gas) in the syngas used (Table 6) could explain this lack of growth of these two strains. Thus, A. malicum and A. woodii would not be able to assimilate CO, under the culture conditions tested.

In contrast, the three new strains tested (P4066, P4073 and P4074) showed significant growth in the presence of syngas as the only source of carbon and energy under the culture conditions tested. These strains are therefore able to use syngas as the only source of carbon and energy and to assimilate CO, under the culture conditions tested, unlike the control strains A. fimetarium, A. malicum and A. woodii.

y) Medium volume culture tests

The 6th subculture (6R) was carried out in a Penicillin bottle (50 mL) in order to assess the consumption and production of gas (H2, CO2 and CO) in micro-GC, and to monitor growth (DOôoo) and pH over a period of at least 3 weeks, in medium-volume crops.

The culture conditions of the strains in Penicillin flasks (6R) are as follows: 50 ml culture medium (Penicillin flask of 121.5 ml)

Inoculation at 10% (v / v)

Incubation at 25 ° C for strains P4066, P4073 and P4074 and at 30 ° C for typical strains.

Orbital shaking: 180 rpm except for P4112, static incubation.

Growth monitoring: D06oo and pH (strip)

GC analyzes: acetate and ethanol production

Micro-GC analyzes: consumption and production of gas (H2, CO2 and CO)

The results of growth monitoring, pH, production or consumption of gas and production of metabolites are presented in Figures 6 to 9.

The new strains P4066, P4073, P4074 and the control strain P4112 (A. wieringae, non-agitated culture) exhibited maximum DO6oo values of between 0.8 and 1 with significant acidification during the kinetics.

The consumption of ΓΗ2 and CO were significant for these 4 strains. In the same way as for the cultures carried out under the conditions used during the isolations (paragraph a above), a production of CO2 is observed for the strains P4066, P4073 and P4074 during the phase of exponential growth, which decreases strongly during of the stationary phase (Figures 6 to 8). On the other hand, the control strain P4112 showed regular and significant production of CO2 until the end of culture. This control strain is therefore not capable of reducing its production of CO2, under the culture conditions tested (Figure 9).

The 4 strains tested produced acetate. However, only the new strains P4066, P4073 and P4074 produced ethanol, unlike the control strain P4112 (A. wieringae). In addition, once the switch from acetate to ethanol production had been made for these strains, the quantities of ethanol produced were significantly greater than the quantities of acetate produced throughout the duration of the stationary phase, up to at the end of culture for 2 of the 3 strains (P4073 and P4074, Figures 7 and 8).

Furthermore, the strain P4109 (A. carbinoculum) did not show significant growth in the tests carried out in the Penicillin bottle, unlike the tests in the Hungate tube. Thus, this strain loses its growth capacity in the presence of syngas during the transition from the small culture scale (5mL, Hungate) to the medium scale (50 mL, Penicillin bottle).

The strains P4066, P4073 and P4074 are physiologically different from the closest phylogenetically type strains because they exhibit significant differences in growth and metabolism in the presence of syngas. Surprisingly, these 3 new isolated strains are all capable of synthesizing ethanol and acetate using syngas as the only substrate, both in small (5mL) and medium (50 mL) volumes. Among the Acetobacterium sp. of the prior art tested, only the A. wieringae (P4112) strain is capable of growing under the same conditions. However, this strain is not capable of producing ethanol in the presence of syngas.

The other Acetobacterium sp. of the tested prior art are not capable of growth under all of the culture conditions tested. Thus, A. fimetarium (P4110), A. malicum DSM 4132 (P4104) and A. woodii DSM 1030 (P4108) are not capable of growth in the presence of the syngas used in this study as the only source of carbon and energy, whatever the conditions tested. The A. carbinoculum strain (P4109) is no longer able to grow in the presence of syngas when the culture volume increases.

This study thus highlights for the first time a group of new strains of the genus Acetobacterium sp., Which are capable both of growing in the presence of syngas composed of a mixture of CO, H2 and CO2 and of using this syngas as a substrate to produce significant amounts of ethanol and acetate. These new strains are original because they are capable of assimilating a substrate containing CO to produce acetate and ethanol, under all the culture conditions tested, unlike the strains Acetobacterium sp. from the prior art.

The new strains thus identified in this study are all the more interesting as they are capable of producing acetate and then ethanol, in a differential manner, thanks to a switch in the production of l acetate to ethanol. Depending on the culture conditions, these new strains are capable of maintaining a high level of ethanol production and a lower, if not almost zero, level of acetate production. These strains therefore offer the possibility of playing on the conditions and the culture time, depending on the final product of desired interest.

The 6 new strains of the genus Acetobacterium sp. identified in this study therefore represent microorganisms of choice for implementing industrial bio-production processes of ethanol and / or acetate, in particular ethanol, from syngas, in particular from syngas containing CO.

BIBLIOGRAPHICAL REFERENCES

Balch WE, Schoberth S, Tanner RS, Wolfe RS, (1977). Acetobacterium, A New Genus of Hydrogen Oxidizing, Carbon Dioxide Reducing, Anaerobic Bacteria. Int. J. Syst. Bacteriol. 27: 355-361.

Bertsch J and Müller V, (2015). CO Metabolism in the Acetogen Acetobacterium woodii. Appl Environ Microbiol. Sep 1; 81 (17): 5949-56.

Buschhorn H, Dürre P, Gottschalk G, (1989). Production and Utilization of Ethanol by the Homoacetogen Acetobacterium woodii. Appl Environ Microbiol. Jul; 55 (7): 1835-1840.

Claims (16)

1. A strain isolated from Acetobacterium sp. Bacteria, characterized in that it has the capacity of a strain chosen from the strains deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, Paris, France), under the registration numbers CNCM-l-5192, CNCM-l-5194, CNCM-l-5195, CNCM-l-5191, CNCM-I-5193, and CNCM-l-5190, to be produced by fermentation of at least one compound d 'industrial interest. 2. Isolated strain according to claim 1, characterized in that it has the capacity of the strain CNCM-l-5192 or of the strain CNCM-l-5194, to produce by fermentation at least one compound of industrial interest. 3. Isolated strain according to claim 1, characterized in that it is chosen from the strains CNCM-l-5192, CNCM-l-5194, CNCM-l-5195, CNCM-l-5191, CNCM-l-5193, and CNCM-l-5190. 4. Isolated strain according to claim 3, characterized in that it is the strain CNCM-l-5192 or the strain CNCM-l-5194. 5. Biologically pure culture of an isolated strain according to any one of claims 1 to 4. 6. Method for producing a compound of industrial interest by fermentation, comprising the steps consisting in: (i) culturing at least one isolated strain according to any one of claims 1 to 4, in the presence of an appropriate substrate , in a culture medium and under conditions allowing the growth of said isolated strain, so as to produce said compound of industrial interest; (ii) optionally recovering the culture medium comprising said compound, produced in step (i); and (iii) optionally, the purification of said compound from the culture medium recovered in step (ii). 7. Method according to claim 6, in which said conditions are fermentation conditions comprising: a temperature between 20 and 37 ° C, preferably between 22 and 35 ° C, more preferably between 24 and 33 ° C , more preferably between 25 and 30 ° C; and / or - agitation; and / or - an anaerobic atmosphere. 8. Method according to claim 6 or 7, in which the culture of step (i) and optionally step (ii) is (are) carried out in a bioreactor in batch mode, in fed-batch mode or in continuous mode. 9. Use of an isolated strain according to any one of claims 1 to 4 to produce a compound of industrial interest. 10. Use according to claim 9, in which said compound of industrial interest is produced by fermentation of a suitable substrate by means of said isolated strain. 11. Method according to any one of claims 6 to 8, or use according to claim 10, wherein said suitable substrate comprises a source of carbon. 12. Method or use according to claim 11, wherein said suitable substrate is in gaseous form. 13. Method or use according to claim 11 or 12, wherein said carbon source is chosen from a sugar, such as glucose, fructose, dextrose; a carboxylic acid or a salt of carboxylic acid, such as acetate, oxaloacetate, formate, fumarate, malate; an alcohol, such as methanol, ethanol, propanol, butanol, pentanol; a diol, such as propane diol, butane diol; ethylene glycol, tetraethylene glycol; polyethylene glycol (PEG); methoxyethanol; ethoxyethanol; tn'methoxybenzoate; tn'methocycinnamate; acetoin; glycerol; lactate; pyruvate; methoxyacetate; glycolate; caffeate; ethanolamine; choline; tn'methylamine; betaine; glycine; the syngas; and any mixture thereof. 14. Method or use according to claim 13, in which said carbon source is syngas, said syngas preferably comprising carbon dioxide (CO2) and dihydrogen (H2), or carbon monoxide (CO) and optionally hydrogen, or a mixture of carbon dioxide, dihydrogen and carbon monoxide. 15. An isolated strain according to claim 1 or 2, method according to any one of claims 6 to 8 and 11 to 14, or use according to any one of claims 9 to 14, wherein said compound of industrial interest is a alcohol, a carboxylic acid, a salt of a carboxylic acid or any mixture thereof. 16. The strain, method or use according to claim 15, wherein said compound of industrial interest is ethanol, acetic acid, a salt of acetic acid, or a mixture thereof, said compound of interest industrial preferably being ethanol.