contaminated with 1 ,2-dichloroethane.

The present invention relates to the sector of environmental safety and more precisely the sector of environmental remediation.

In particular, the invention relates to a consortium of microorganisms useful for the bioremediation of bodies of water contaminated with 1 ,2-dichloroethane (1 ,2-DCA).

Even more preferably, the present invention relates to a consortium of microorganisms that can degrade 1 ,2-DCA to ethylene, substantially without producing toxic halogenated intermediates, said consortium, when present in an aqueous liquid, preferably under anaerobic conditions, being tolerant to a concentration of 1 ,2-DCA in solution in said aqueous liquid greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L, and more preferably comprised between 2000 mg/L and 4000 mg/L. In a second aspect, the present invention relates to a method for the bioremediation of a body of water contaminated by 1 ,2-DCA, where in said body of water the concentration of 1 ,2-DCA in solution may be greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L and more preferably comprised between 2000 mg/L and 4000 mg/L, which comprises placing in contact with said body of water, preferably under anaerobic conditions, the aforementioned consortium of microorganisms able to degrade the 1 ,2-DCA.

The aforementioned method may be used, for example, for the bioremediation of confined or unconfined aquifers, aquatic sediments, submerged soils, water tables, and may be applied both on site, i.e. to the water extracted from said body of water and in situ, i.e. directly to the body of water, and preferably it is performed in situ. 1 ,2-DCA is harmful to humans and animals, is an irritant to the respiratory tracts, highly flammable, and is a possible carcinogenic agent (group 2B of the International Agency for Research on Cancer, IARC).

The main use of 1 ,2-DCA is in the plastic industry, as an intermediate in the synthesis of vinyl chloride, in turn a precursor of polyvinyl chloride (PVC). Accidental spills and the negligent management of the production, storage, transport and use processes in the aforementioned industrial sector, have contributed to the spread of significant quantities of this hazardous pollutant in the environment. In particular, the water solubility and high chemical stability (the half-life in anaerobic aquifers is about 50 years) of 1 ,2-DCA, make it a "perennial" pollutant of aquifers, with a very high risk to the environment and to human health.

Conventionally, soils contaminated with persistent pollutants may be treated ex situ, i.e. removed and subjected to chemical/physical treatments such as, for example, washing with solvents, emulsifiers and detergents, vapor at high temperature, etc.

In particular, for the chemical/physical treatment of contaminated bodies of water it is conventional to resort to extraction techniques with solvents, thermal desorption and stripping of pollutants, preferably coupled with adsorption processes on solid matrices (e.g. active carbon, functionalized zeolites, etc.), in order to make the aforementioned pollutants available for chemical or physical degradation. However, these treatments are very expensive and energy intensive, and also pose the problem of the disposal of the contaminated adsorbent materials.

An advantageous alternative is represented by in situ bioremediation methods, which exploit the degradation activity of autochthonous and/or exogenous microorganisms, promoting the transformation of toxic organic compounds into harmless substances such as, for example, carbon dioxide, methane, water, inorganic salts and the like.

It is however important to note that the family of toxic organochlorinated compounds persisting in the environment is very extensive and comprises compounds that are very different from one another.

In general, chlorinated hydrocarbons having a higher number of chlorine atoms are generally recalcitrant to aerobic degradation, while they are commonly biotransformed in conditions with a lack of oxygen.

Over recent years, research into dehalogenation by anaerobic microorganisms has been particularly intense. Numerous species of the Dehalococcoides, Dehalobacter,

Desulfitobacterium and Dehalogenimonas genera have been shown to be able to couple the reductive dehalogenation of organohalogenated compounds with the conservation of energy and growth, through a process defined as OrganoHalide Respiration (OHR), and for some of these it has been possible to identify the enzymes and isolate the genes involved in this particular metabolic route.

These anaerobic microorganisms, which exploit sources of carbon in the reduced state (e.g. lactate, succinate, pyruvate, benzoate, propionate or formiate) and/or hydrogen as electron donors and are able to use the halogenated compounds as final electron acceptors in anaerobic respiration (Smidt, H. and de Vos, W.M. "Anaerobic microbial dehalogenation" (2004), Annu. Rev. Microbiol., vol. 58, pag. 43-73) are therefore the best candidates for in situ bioremediation treatments.

It is necessary to consider that for some species that are particularly recalcitrant to in vitro cultures and requiring conditions of total absence of oxygen, the study of enzyme activity associated with reductive dehalogenation has been shown to be particularly challenging and has required the use of innovative "ecogenomics" techniques, as described, for example, in F. Maphosa et al., "Ecogenomics of microbial communities in bioremediation of chlorinated contaminated sites" (2012) Front. Microbiol., vol. 3, art. 351.

It is also important to note that the degradation of organohalogenated compounds may lead to the formation of intermediates that are just as harmful or even more harmful than the starting substrates, (for example trichloroethane (TCE) and tetrachloroethane (PCE) are transformed into vinyl chloride), actually limiting the application of these

microorganisms for in situ bioremediation.

Despite this, numerous bioremediation methods for aquifers and soils contaminated by chlorinated alkenes or alkanes via microbiological methods are described in the known art.

For example, EP 0 864 542 A2 describes a method for the degradation of chlorinated ethylenes and propylenes present in ground water, which is conveyed, after the addition of an electron donor compound, into a reactor containing a microbial consortium able to degrade halogenated alkenes under anaerobic conditions.

US 6,001 ,252 on the other hand describes a method for the in situ bioremediation of aquifers which comprises the inoculation in the contaminated site of a bacterial consortium able to promote the dehalogenation of the solvents present under anaerobic conditions.

Bioremediation interventions may also comprise the "biostimulation" of degrading microorganisms already present in the contaminated site, providing them with the essential compounds (e.g. nutrients, gases, sources of carbon, mineral salts, etc.) for the purpose of making them prosper and making them able to efficiently perform the task of decomposing the pollutants.

For example, EP 1 600 220 A1 describes an "additive" for use in the bioremediation of soils, water tables or sediments contaminated by organic halides, which comprises a material for developing and activating a microbial consortium comprising active yeast, inactive yeast and yeast extract, a material for promoting reductive dehalogenation by the anaerobic microorganisms, which comprises a compound selected from propionic acid, butyric acid, lactic acid and salts thereof and a material for developing anaerobic conditions, which comprises a compound selected from glucose, galactose, fructose, lactose, sucrose, maltose and starch.

Furthermore, bioremediation strategies may include "bioaugmentation" interventions, through which certain bacterial species with excellent degrading abilities are introduced into contaminated sites, with the aim of supporting or substituting the activity of the autochthonous species when the latter alone are not shown to be able to guarantee a significant bioremediation process in reasonable time frames.

For example, WO 95/32278 describes a method for the bioremediation of soil

contaminated by polycyclic aromatic hydrocarbons, comprising the introduction into the soil of a mixed bacterial culture of Acrhromonas sp. and Mycobacterium sp., previously accustomed to the polycyclic aromatic hydrocarbons, together with a system of nutrients containing a source of nitrogen and a source of phosphate in order to support the growth of said microorganisms.

In none of the above cases is the bioremediation method applied to sites contaminated by one of the most important and dangerous chlorinated compounds, i.e. 1 ,2-DCA.

EP 1 352 977 A2 describes a bacterial strain belonging to the Desulfitobacterium genus able to dehalogenate dichloroalkanes and trichloroalkanes with high conversion speeds, possibly in the presence of bacterial species able to produce vitamin K, such as,

Enterococcus sp. It is important to note that the strain described in EP 1 352 977 A2 was isolated from a layer of soil contaminated by 1 ,2-DCA in a concentration equal to 50 mg/kg and its dehalogenating activity was measured using concentrations of

dichloroalkanes and trichloroalkanes no greater than 400 μΜ.

Similarly, US 8,673,614 describes a microbial consortium comprising strains of the Clostridium, Acetobacter, Dehalobacter, Bacterioides, Proteobacteria and

Dehalococcoides genera, isolated from samples of sediment from a site contaminated by chloroethanes and chloroethylenes in an anaerobic medium containing chloroethane and an electron donor compound selected from lactate, succinate, pyruvate, benzoate, propionate or formiate. Also in this case the concentration of chlorinated organic compounds is never greater than 50 μΜ.

Finally, patent JP 5764880 B2 describes a strain of Geobacter sp. able to transform 1 ,2- DCA to ethylene through an anaerobic dehalogenation reaction, although not displaying any degradation activity on chlorinated pollutants such as tetrachloroethylene, 1 ,1 ,2- trichloroethane, 2,4,6-trichlorophenol, I'hexachlorobenzene. The aforementioned strain of Geobacter sp., whose activity was measured on 1 ,2-DCA in concentrations no greater than 1 mM, was used in presence of other bacterial species that act on other pollutants (for example, able to dehalogenate vinyl chloride), for the optimization of the

bioremediation capacity.

However, the known solutions still have margins for improvement.

In general, many environmental factors, such as the lack of nutritional factors, non-optimal pH and/or redox potential values, the presence or absence of oxygen, may reduce the degrading capacity of the microorganisms. Autochthonous bacterial species or those introduced through bioaugmentation interventions, implied in bioremediation processes for contaminated aquifers may die in presence of chlorinated organic compounds; as described, for example, by J.C. Koenig et al. in "Tolerance of Anaerobic Bacteria to Chlorinated Solvent" (2014), Microbes Environ., vol. 29, pag. 23-30, a significant limitation to the bioremediation of sites contaminated by chlorinated organic compounds actually consists of the onset of toxic effects by said compounds on the microorganisms, especially due to damage to the bacterial membranes connected with the solvent properties for the lipids of these compounds. In this context, the type of chlorinated organic compounds is important, since some of these have been shown to be more recalcitrant to the degradation performed by the microorganisms.

For example, the presence of 1 ,2-DCA may inhibit the degradation of other pollutants, both halogenated and non-halogenated (as described, for example, by B.A. Kocamemi et al. in "Inhibitory effect for the xenobiotic 1 ,2-dichloroethane in a nitrifying biofilm reactor" (2007), Water Sci. Technol., vol. 55, pag. 67-73 and by K. Mayer-Blackwell et al., in "1 ,2- Dichloroethane Exposure Alters the Population Structure, Metabolism, and Kinetics of a Trichloroethene-Dechlorinating Dehalococcoides mccartyi Consortium" (2016), Environ. Sci. Technol., vol. 50, pag. 12187-12196), compromising the efficacy of the

bioremediation intervention.

For this reason, most of the reductive dehalogenation studies of 1 ,2-DCA by microbial consortia have been performed using decisively reduced concentrations of said pollutant (for example, from 0.1 to 0.5 mM), and the most resistant species described in literature have shown dehalogenating activity, although not optimal, up to concentrations equal to 8.7 mM of 1 ,2-DCA (A.D. Maness et al., "Dehalogenimonas spp. can reductively dehalogenate high concentrations of 1 ,2-dichloroethane, 1 ,2-dichloropropane, and 1 ,1 ,2- trichloroethane" (2012) AMB Express, vol. 2, art. 54).

However, there are numerous contaminated sites in which the quantities of these contaminants may be substantially higher (for example M. Marzorati et al. in "A novel reductive dehalogenase, identified in a contaminated groundwater enrichment culture and in Desulfitobacterium dichloroeliminans strain DCA1 , is linked to dehalogenation of 1 ,2- dichloroethane" (2007) Appl. Environ. Microbiol, vol. 73, pag. 2990-2999 describes an aquifer contaminated with high concentrations, up to 9 mM, of 1 ,2-DCA).

There is therefore a strongly felt need to be able to have a method available for treating a body of water contaminated by chlorinated organic compounds, in particular contaminated by 1 ,2-DCA, in particular when the concentration of this contaminant is high, which can overcome the drawbacks highlighted in methods of the prior art.

The Applicant has now identified a consortium of microorganisms able to promote the degradation of 1 ,2-DCA to ethylene, preferably under anaerobic conditions, without producing toxic halogenated intermediates, efficiently, said consortium, when present in an aqueous liquid, also being tolerant to high concentrations of said contaminant, unexpectedly significantly higher with respect to the tolerated concentrations for adaptation and typically found in the prior art.

The aforementioned consortium comprises the microorganisms deposited on 8 November 2016 in accordance with the Budapest Treaty on the "National Collection of Industrial Food and Marine Bacteria" (NCIMB), Aberdeen, Scotland, UK, to which accession number NCIMB 42687 was assigned.

In particular, the degrading activity on the 1 ,2-DCA substrate by the aforementioned consortium is also displayed, preferably under anaerobic conditions, when such pollutant is present in solution in said aqueous liquid at concentrations greater than or equal to 1000 mg/L and comprised between 1000 mg/L and 4000 mg/L, even more preferably comprised between 2000 mg/L and 4000 mg/L. The Applicant has also identified a method for the bioremediation of a body of water contaminated by high concentrations of 1 ,2-DCA in solution, preferably under anaerobic conditions, which uses the aforementioned consortium of microorganisms. Preferably, said method is performed in situ, i.e. without it being necessary to extract the water from said body of water.

Thanks to the ability of the aforementioned consortium to maintain the dehalogenating activity, preferably under anaerobic conditions, in presence of high concentrations of 1 ,2- DCA, preferably at a concentration greater than or equal to 1000 mg/l, more preferably comprised between 1000 mg/l and 4000 mg/l, even more preferably comprised between 2000 mg/l and 4000 mg/l, the method according to the present invention has the advantage of being able to guarantee the bioremediation of a body of water or an aquifer wherein the concentration of said pollutant in solution can reach and exceed the maximum concentration values of said pollutant that can be treated with the bioremediation methods described in the art.

In particular, with the method according to the present invention, it is possible to bioremediate, preferably under anaerobic conditions, a body of water contaminated by 1 ,2-DCA wherein the concentration of said pollutant in solution may be greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L, more preferably comprised between 2000 mg/L and 4000 mg/L.

With the method according to the present invention, the body of water contaminated by 1 ,2-DCA can be treated in situ: in other words, the bioremediation of the polluted water may be performed without it having to be taken from the body of water. Despite this, the aforementioned method is also suitable for on-site treatments, i.e. on the water extracted from the body of water and placed in contact with the dehalogenating consortium in a second confined space, such as a cistern, a tank, a bath, a reactor, etc. The

bioremediation of the body of water with the consortium NCIMB 42687 is preferably performed under anaerobic conditions.

It is important to note that the method according to the present invention may obtain public acceptance without difficulties both due to its beneficial effect on the environment, connected with the dehalogenation activity expressed by the consortium NCIMB 42687 on the pollutant 1 ,2-DCA, and due to the fact that said consortium does not comprise microorganisms manipulated with genetic engineering techniques.

Further characteristics and advantages of the present invention will become clear from the following detailed description.

For the purposes of the present description and following claims, the definitions of the numeric ranges always include the extremes unless specified otherwise.

In the description of the embodiments of the present invention, the use of the terms "comprising" and "containing" indicates that the options described, for example regarding the steps of a method or of a process or the components of a product or of a device, are not necessarily all-inclusive. It is however important to note that the present application also relates to the embodiments in which the term "comprising" in relation to the options described, e.g. regarding the steps of a method or of a process or the components of a product or of a device, must be interpreted as "which essentially consists of" or "which consists of, even if this is not explicitly stated.

For the purposes of the present description and following claims, "ecogenomics" means a range of technologies that do not require the isolation and reproduction of the

microorganisms but are applied directly to environmental samples, comprising whole genome amplification, "high-throughput" sequencing of the DNA ("metagenomics") and the RNA ("metatranscriptomics"), the identification of peptides ("metaproteomics") and metabolites ("metabolomics") through mass spectrometry, and other sophisticated techniques that are combined according to new modelling approaches and thanks to computational analysis, in an integrated ecological framework.

For the purposes of the present description and following claims, the term "consortium" of microorganisms means a mixed culture of two or more species of microorganisms, where the two or more species can live in symbiosis and/or be involved at least in part in one or more biological processes where each species can take advantage from the other components of the consortium.

The mixed culture in an ideal case may be syntropic, i.e. characterized by microorganisms all operating in the same biological process, such as the bioremediation of soils, in an ordered and synergistic way towards a single aim, such as, the bioremediation of polluting compounds, and/or syntrophic, i.e. characterized by species of microorganisms that prosper at the expense of the metabolites produced by other species, in an

interdependent relationship.

For the purposes of the present description and following claims, the terms "tolerance to 1 ,2-DCA", or simply "tolerance", used to describe a microorganism or a consortium of microorganisms, means the ability of a microorganism or a consortium of microorganisms to survive and multiply in an aqueous liquid, for example in a liquid or aquiferous medium in which 1 ,2-DCA is present in a concentration greater than or equal to 1000 mg/L, preferably in a concentration comprised between 1000 mg/L and 4000 mg/L and more preferably in a concentration comprised between 2000 mg/L and 4000 mg/L. When the microorganism is anaerobic or the consortium of microorganisms comprises at least one anaerobic microorganism, the aforementioned "tolerance" is generally preferably displayed when said microorganism or said consortium is kept under anaerobic conditions.

For the purposes of the present description and the following claims, "sequence identity percentage (%)" referring to a nucleotide or amino acid sequence is defined as the percentage of nucleotides or of amino acids in a nucleotide or amino acid sequence being examined that are identical to the nucleotides or to the amino acids of a reference nucleotide or amino acid sequence. The sequence identity can be determined using programs that are known to a person skilled in the art, such as BLAST, ALIGN and CLUSTAL, using standard methods (for example as described by S. F. Altschul, et al. in "Basic Local Alignment Search Tool" (1990), J. Mol. Biol. vol. 215, pag. 403-410, by S. Henikoff et al. in "Amino Acid Substitution Matrices from Protein Blocks" (1992), Proc. Natl. Acad Sci. USA, vol. 89, pag. 10915-10919, by S. Karlin et al. in "Applications and Statistics for Multiple High-Scoring Segments in Molecular Sequences" (1993), Proc. Natl Acad. Sci USA, vol. 90, pag. 5873-5877 and by D.G. Higgins et al. in "CLUSTAL: a Package for Performing Multiple Sequence Alignment on a Microcomputer" (1988), Gene, vol. 73, pag.237-244. The software for performing BLAST analyses is made available to the public by the National Center for Biotechnology Information (NCBI). Furthermore, it is possible to perform research in the sequence databases using the program FASTA (W.R. Pearson et al., "Improved Tools for Biological Sequence Comparison" (1988), Proc. Natl. Acad. Sci. USA, vol. 85, pag. 2444-2448).

An indication that two sequences of amino acids (polypeptides) are substantially identical is given by the immunological cross-reactivity between the two polypeptides. Generally, polypeptides that differ from one another through conservative substitutions of amino acids, i.e. in which one amino acid is substituted with another characterized by a lateral group with similar biochemical properties, have immunological cross-reactivity. Therefore, a polypeptide is substantially identical to a second polypeptide, for example, when the two polypeptides differ from one another by conservative substitution of amino acids. An indication that two nucleotide sequences are substantially identical (they have a sequence identity of 97% or greater than 97%) is that the two molecules (one of which is defined as the "probe") hybridize together under stringent conditions (e.g. in a medium-high

"stringency" range). "Hybridization conditions" means the conditions in which the hybridization reactions are performed. These conditions are generally classified based on the degree of "stringency" of the conditions with which the hybridization is measured. The degree of "stringency" can be based, for example, on the difference between the hybridization temperature and the Melting Temperature, T _m , of the complex formed by the nucleotide sequence being examined and by the "probe" sequence. Alternatively, or additionally, the degree of stringency can be based on the ionic force conditions of the solutions in which the hybridization experiments are performed.

Functionally, the hybridization of a polynucleotide with a probe module in maximum stringency conditions corresponds to a total or almost total sequence identity between the two sequences.

For the purpose of better understanding the characteristics of the process according to the present invention, reference shall also be made to the drawings of the appended figures that have a purely illustrative and absolutely non-limiting function.

In particular, Figure 1 schematically represents the genome fragment of Geobacter sp. which represents the region of DNA in which the genes associated with the enzyme 1 ,2- dichloroethane dehalogenase are present. The enzyme activity is typically attributed to the set of proteins codified by the gene rdhA and rdhB. In the aforementioned DNA region, there are homologous sequences to the "Two component system response regulator ¹ ' (5' end) and to the emrB/qacA (3' end) genes of Geobacter sp.

Figure 2 represents a graph showing the reduction, over nine years, of the concentration of 1 ,2-DCA in solution in the water contained in a piezometric well placed in an industrial site contaminated by 1 ,2-DCA, and from which the consortium NCIMB 42687 was isolated.

Figure 3 is a graph showing the percentage reduction of the concentration of 1 ,2-DCA (Figure 3a) and a graph showing the percentage increase of ethylene production (Figure 3b) in microcosms in which the consortium NCIMB 42687 is cultivated in presence of concentrations of 1 ,2-DCA equal to 50 mg/L, 300 mg/L, 1000 mg/L, 2000 mg/L and 4000 mg/L. The quantity of ethylene is represented as the area under the peak of the gas chromatogram associated with such compound.

Figure 4 represents a graph showing the reduction of the concentration of 1 ,2-DCA in the aforementioned piezometric well over the course of the experiment described in Example 4.

It is important to note that the drawings of the appended figures intend to illustrate the general characteristics of the method and/or the materials used in some embodiments of the invention and to complete the written description below.

These drawings do not necessarily accurately reflect the precise structural characteristics or performance of a given embodiment and should not be interpreted as defining or limiting an interval of values or properties of said embodiment.

The present invention relates firstly to a consortium of microorganisms, deposited on 8 November 2016 in accordance with the Budapest Treaty at the "National Collection of Industrial Food and Marine Bacteria" (NCIMB), Aberdeen, Scotland, UK, to which accession number NCIMB 42687 was assigned. The aforementioned consortium NCIMB 42687 can degrade, preferably under anaerobic conditions, the 1 ,2-DCA to ethylene, substantially without producing toxic halogenated intermediates and can be tolerant to a concentration of 1 ,2-DCA greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L, and more preferably comprised between 2000 mg/L and 4000 mg/L.

The concentration of 1 ,2-DCA relates to the quantity of said contaminant in solution per unit of volume, for example, in an aqueous liquid in which the aforementioned consortium is present and in which it is, for example, maintained or cultivated.

The consortium of the present invention comprises both obligate anaerobic species of microorganisms and oxygen tolerant species. For this reason, all the operations relating to said consortium are performed so as to reduce, as much as possible, contact with the atmospheric oxygen and/or that dissolved in an aqueous liquid.

The aforementioned consortium NCIMB 42687 was isolated starting from samples of water coming from an industrial site polluted by 1 ,2-DCA, and is characterized by stable and constant reductive dehalogenation activity on the aforementioned chlorinated hydrocarbon and tolerance to 1 ,2-DCA in particularly high concentrations, as specified above.

The reductive dehalogenation activity characteristic of the NCIMB consortium may be responsible for the degradation of 1 ,2-DCA by the aforementioned consortium and for the production of ethylene.

The degradation of the 1 ,2-DCA, and the concomitant production of ethylene, by the consortium NCIMB 42687 may be associated with the reductive dehalogenation activity by at least one 1 ,2-dichloroethane dehalogenase enzyme synthesized by at least one microbial species of the consortium.

In a preferred aspect, said consortium of microorganisms can synthesize at least one 1 ,2- dichloroethane dehalogenase enzyme having an amino acid sequence that has a sequence identity equal to 97% or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 6. More preferably, said consortium of

microorganisms can synthesize at least one 1 ,2-dichloroethane dehalogenase enzyme having an identical amino acid sequence to the sequence SEQ ID NO: 6.

To determine the identifying characteristics of its components, the consortium NCIMB 42687 was subjected to the analysis of the ribosomal DNA (16S rDNA profiling) through metagenomic sequencing using Ion Torrent™ technology, following the protocols provided by the manufacturer, as described in Example 2, through which it was possible to isolate and characterize four different sequences of 16S rDNA, representing the preponderant species of microorganisms in said consortium, which correspond to the sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4.

Based on the comparison with the sequences present in the reference databases using the program BLAST ^® (NCBI), it was possible to attribute the four different sequences of 16S rDNA to the species: Geobacter sp. (SEQ ID NO: 1 ), Pseudomonas sp. (SEQ ID NO: 2), Clostridium sp. (SEQ ID NO: 3) and Acidovorax sp. (SEQ ID NO: 4).

In other words, based on the analysis of the 16S ribosomal DNA it is considered that the anaerobic consortium NCIMB 42687 may preponderantly comprise at least one strain of the species Geobacter sp., at least one strain of the species Pseudomonas sp., at least one strain of the species Clostridium sp. and at least one strain of the species Acidovorax sp. The aforementioned consortium may comprise further species of anaerobic or oxygen- tolerant microorganisms, with respect to those indicated above, which cannot however be identified either through the traditional microbiological techniques of bacterial isolation known to a person skilled in the art or through the metagenomic analyses described above, therefore they are not considered significant for the purposes of the present invention.

Preferably, the consortium may comprise microorganisms characterized by at least one nucleotide sequence that has a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 1 , or with the sequence SEQ ID NO: 2, or with the sequence SEQ ID NO: 3, or with the sequence SEQ ID NO: 4, or by combinations of nucleotide sequences, each of which has a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO:1 , or with the sequence SEQ ID NO: 2, or with the sequence SEQ ID NO: 3, or with the sequence SEQ ID NO: 4.

In a particularly preferred aspect the consortium of microorganisms NCIMB 42687 may comprise at least one strain of at least one species of Geobacter sp. having an identical 16S rDNA nucleotide sequence to the nucleotide sequence SEQ ID NO: 1 , at least one strain of at least one species of Pseudomonas sp. having an identical 16S rDNA nucleotide sequence to the nucleotide sequence SEQ ID NO: 2, at least one strain of at least one species of Clostridium sp. having an identical 16S rDNA nucleotide sequence to the nucleotide sequence SEQ ID NO: 3, at least one strain of at least one species of Acidovorax sp. having an identical 16S rDNA nucleotide sequence to the nucleotide sequence SEQ ID NO: 4.

From the analyses of the genomic DNA extracted from samples of the consortium NCIMB 42687 it can further be demonstrated that the reductive dehalogenation activity displayed by the aforementioned consortium is connected with the abundance of 16S rDNA homologous to the sequence SEQ ID NO: 1 . For this reason, it is reasonable to suppose that the enzyme responsible for such reductive dehalogenation activity is synthesized by at least one strain of the species associated with the sequence SEQ ID NO: 1 , i.e.

Geobacter sp. This hypothesis is confirmed by the result of the sequencing of the metagenome of the consortium, as described better below.

Therefore, in a preferred aspect, the consortium NCIMB 42687 comprises at least one strain of at least one species of Geobacter sp. having a 16S rDNA nucleotide sequence that may have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99%, or is identical to the nucleotide sequence SEQ ID NO: 1 , in whose genomic DNA there may be at least one rdhA gene coding for the 1 ,2- dichloroethane dehalogenase enzyme.

Preferably, said rdhA gene coding for the 1 ,2-dichloroethane dehalogenase enzyme may have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 5. Preferably, said rdhA gene has an identical nucleotide sequence to the sequence SEQ ID NO: 5.

Preferably, said rdhA gene codes for a protein of 551 amino acids that may have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 6. Preferably, said rdhA gene codes for a protein having an identical amino acid sequence to the sequence SEQ ID NO: 6.

The metagenomic sequencing using Ion Torrent™ technology and following the protocols provided by the manufacturer starting from samples of said anaerobic consortium NCIMB 42687 has demonstrated that the aforementioned rdhA gene is present in a region of about 4000 pairs of bases, which may comprise four genes that have a high homology with the four genes rdhA, rdhB, rdhC and rdhT, described, for example, by Futagami T., Goto M., Furukawa K. in "Biochemical and genetic bases of dehalorespiration" (2008) Chem Record, vol. 8, pag. 1 -12.

Said region of genomic DNA is represented schematically in Figure 1 .

The analysis of the nucleotide sequences both upstream and downstream of the aforementioned genomic DNA of about 4000 pairs of bases has enabled sequences to be identified which, from the analysis of the homology with sequences contained in the databases consulted appear to be characteristic of the species Geobacter sp. and univocally attributable to such species. This confirms the hypothesis that the rdhA gene may be present in the genomic DNA of the species Geobacter sp.

In a preferred aspect of the invention, the aforementioned rdhA gene may be comprised in a genomic DNA region that can comprise a sequence attributable to the rdhB gene, which codes for an anchoring protein to the membrane associated with the dehalogenating enzyme, having a sequence that can have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 7 and more preferably may have an identical sequence to the sequence SEQ ID NO: 7.

In a preferred aspect of the invention, the aforementioned rdhA gene may be comprised in a genomic DNA region that may comprise a sequence attributable to the rdhC gene, coding for a protein probably with a transcriptional regulation function of the rhdA gene, having a sequence that may have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 8 and more preferably may have an identical sequence to the sequence SEQ ID NO: 8.

In a preferred aspect of the invention, the aforementioned rdhA gene may be comprised in a genomic DNA region that may comprise a sequence attributable to the rdhT gene, coding for a protein probably involved in the correct folding in the presence of a corrinoid factor of the precursor of the dehalogenating enzyme, having a sequence that may have a sequence identity equal to 97%, or greater than 97%, or greater than 98%, or greater than 99% with the sequence SEQ ID NO: 9 and more preferably may have an identical sequence to the sequence SEQ ID NO: 9.

Preferably, the rdhA gene may be present in the genomic DNA of the Geobacter sp. species in combination with at least one of the sequences having sequence identities equal to 97%, or greater than 97%, or greater than 98%, or greater than 99%, or identical to the sequences SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

As mentioned above, the consortium NCIMB 42687 was isolated from samples of water taken from a piezometric well placed in the aforementioned industrial site at the end of a monitoring campaign of the concentration of 1 ,2-DCA, performed from 2004 until 2013. The data related to the concentration of 1 ,2-DCA in solution measured during the aforementioned monitoring campaign are shown in the graph of Figure 2. In particular, it is to be noted that the concentration of pollutant varies over time. Despite a certain fluctuation being highlighted in the dispersion of the data, typical of an unconfined system and connected with phenomena of spreading from and towards the surrounding soil, from the trend line it is possible to demonstrate that the concentration of 1 ,2-DCA in solution is sensitively reduced over time, showing that the consortium effectively displays

degradation activity against 1 ,2-DCA in situ.

The evidence that such degradation activity is associated with the consortium according to the present invention was provided by the results of the analysis of the microorganisms present in samples taken during the aforementioned campaign through extraction of genomic DNA, amplification and sequencing of fragments separated with Denaturing Gradient Gel Electrophoresis (DGGE) techniques. Since 2005, when the first analyses of this type were performed, the composition of bacterial species has been kept constant and in particular it has always been possible to demonstrate in the water samples coming from the aforementioned piezometric well the presence of microorganisms of the

Geobacter sp. species.

Through experiments in microcosms, the consortium NCIMB 42687 has been shown to be effectively able to degrade the 1 ,2-DCA to ethylene substantially without producing toxic halogenated intermediates, preferably under anaerobic conditions, and is therefore particularly suitable for the bioremediation of bodies of water contaminated by said chlorinated hydrocarbon.

In particular, said consortium can degrade the 1 ,2-DCA through reductive dehalogenation even when said pollutant is present in particularly high concentrations in solution and, in fact, it can be maintained in culture in microcosms comprising an aqueous liquid in which 1 ,2-DCA is present in solution in a concentration greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L and more preferably comprised between 2000 mg/L and 4000 mg/L, as it also appears to be vital, able to perform dehalogenation activity, and can therefore degrade the 1 ,2-DCA in bodies of water in which the concentration of 1 ,2-DCA in solution may be greater than or equal to 4000 mg/L, as is clear in Figure 2.

Since within the consortium NCIMB 42687 a species that expresses dehalogenating enzyme activity at high concentrations of 1 ,2-DCA is Geobacter sp., it follows that said species of Geobacter sp. present in said consortium is at least one of the species that are tolerant to 1 ,2-DCA.

In other words, the consortium NCIMB 42687 is characterized by the fact that it comprises at least one strain of a species of Geobacter sp., which displays dehalogenating activity on 1 ,2-DCA and which may be tolerant to a concentration of said 1 ,2-DCA in solution greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L and more preferably comprised between 2000 mg/L and 4000 mg/L.

The consortium NCI MB 42687 may preferably be maintained or cultivated in a synthetic medium, preferably under anaerobic conditions. The aforementioned synthetic medium may be liquid or solid and is preferably liquid.

Therefore, in a preferred aspect, the present invention relates to a culture of said consortium in a synthetic medium, preferably liquid synthetic medium and preferably under anaerobic conditions. Preferably, said synthetic medium comprises at least one electron donor compound, for example an organic acid or a salt thereof, an alcohol, a sugar, or another substrate based on carbon, which simulates the dehalogenation activity of the microorganisms of the consortium and promotes the establishment of anaerobiosis. Preferably, said at least one electron donor compound is selected from the group comprising lactic acid, succinic acid, pyruvic acid, benzoic acid, propionic acid, formic acid and salts thereof with an alkaline metal or alkaline earth metal. In a particularly preferred aspect, said electron donor compound is sodium lactate.

The present invention secondly relates to a method for the bioremediation of a body of water contaminated by 1 ,2-DCA, where in said body of water the concentration of 1 ,2- DCA in solution in said body of water may be greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L and more preferably comprised between 2000 mg/L and 4000 mg/L, which comprises placing in contact with said body of water the consortium NCIMB 42687, preferably under anaerobic conditions.

Preferably, said body of water may comprise, in addition to the 1 ,2-DCA, one or more halogenated aromatic or aliphatic hydrocarbons in solution, for example tetrachloroethylene (PCE) or trichloroethylene (TCE).

In a preferred aspect, the microorganisms of the consortium NCIMB 42687 used in the bioremediation method according to the invention may be in the immobilized form, in accordance with the technologies known to a person skilled in the art. For example, the microorganisms can be immobilized through adhesion to organic or inorganic supports, such as activated carbon or glass, or through incorporation in natural or artificial polymer matrices, for example agarose, gelatin, cellulose, alginate, polyurethane, polyester, etc. In a preferred aspect, the aforementioned method for the bioremediation of a body of water contaminated by 1 ,2-DCA according to the present invention, preferably performed under anaerobic conditions, may comprise the steps of:

a) providing a culture of the enzyme NCIMB 42687 in a synthetic medium containing 1 ,2-DCA in a concentration greater than or equal to 1000 mg/L, preferably comprised between 1000 mg/L and 4000 mg/L, more preferably comprised between 2000 mg/L and 4000 mg/L;

b) placing said culture of the consortium NCIMB 42687 obtained in step a) in contact with the aforementioned body of water.

Step a) of the present method may be performed by growing the aforementioned consortium in any receptacle, for example, microcosm bottles, bioreactors, columns filled with synthetic medium, in accordance with the methods known to a person skilled in the art in which it is possible to recreate an anaerobic environment.

Preferably, the culture of the consortium is created using a synthetic medium based on mineral salts, such as the minimal medium BTZ-3 described by E.R. Hendrickson et al. in "Molecular Analysis of Dehalococcoides 16S Ribosomal DNA from Chloroethene- Contaminated Sites throughout North America and Europe" (2002), Appl. Environ. Microbiol., vol. 68, pag. 485-495, enriched with cysteine, vitamins, and trace metal salts, having a pH comprised between 6.5 and 7.5, as described in Example 1.

Preferably, the culture is performed under anaerobic conditions. To create the anaerobic condition, the culture medium may be subjected to degassing and/or insufflation of inert gases, such as nitrogen, helium, argon, and preferably nitrogen.

In a preferred aspect, in the synthetic medium used for the preparation of the consortium culture of step a) at least one electron donor compound may also be comprised, for example an organic acid or a salt thereof, an alcohol, a sugar, or another substrate based on carbon, which simulates the dehalogenation activity of the microorganisms and promotes the establishment of anaerobiosis.

More preferably, said at least one electron donor compound is selected from the group comprising lactic acid, succinic acid, pyruvic acid, benzoic acid, propionic acid, formic acid and salts thereof with an alkaline metal or alkaline earth metal. In a particularly preferred aspect, said electron donor compound is sodium lactate.

Preferably, said culture provided in step a) is created at a temperature comprised between 15°C and 30°C and more preferably at a temperature comprised between 20°C and 26°C. The duration of step a) may depend on the volume of synthetic medium used and the quantity of microorganisms belonging to the desired consortium, and generally is such as to allow a culture to be obtained in which said microorganisms of the consortium are in the exponential or late exponential phase of growth.

Step b) of the present method may be performed in different ways, according to whether the treatment is conducted in situ or on site. For example, said step b) may be conducted by pumping or injecting the culture obtained in step a) directly into the body of water contaminated by 1 ,2-DCA, through one or more injection points. The methods with which the consortium culture is placed in contact with the body of water contaminated by 1 ,2-DCA may be similar, but not limited, to those described in the prior art, for example, in "Demonstration of Bioaugmentation at Kelly AFB, Texas (2007), ESTCP Cost and Performance Report Environmental Security Technology Certification Program, U.S. Department of Defence (ER-9914), or as described by C.E. Aziz,

R.A.Wymore, R.J.Steffan in "Bioaugmentation - Considerations in Bioaugmentation for Groundwater Remediation" (2013) H.F.Stroo, A. Leeson, C.H.Ward Editors, Springer New York Press.

The volume of culture to be used in step b) may be determined by a person skilled in the art on an individual basis, so as to obtain in the body of water a sufficient quantity of microorganisms of the consortium to guarantee that the consortium itself is kept alive and its propagation. Generally, the volume of culture used is such as not to determine the substantial dilution of the pollutant in the body of water. Furthermore, the ratio between the volume of water to be treated and the volume of culture may increase or decrease based on the growth rate of the consortium in the culture obtained in step a) and the characteristics of the water to be treated, such as its pH, the level of nutrients and electron donor compounds and the concentration of dissolved oxygen. When the method is performed in situ, the aforementioned ratio may also depend on the groundwater flow, the nature and degree of permeability of the soil, and all the other factors that may affect the capacity of the culture of microorganisms to be distributed uniformly and to propagate in the body of water.

As the results of the campaign described above, and presented in Figure 2, show, when the quantity of microorganisms of the consortium used in step b) is adequate, the species of microorganisms of said consortium multiply at the expense of the nutrients present in the body of water and maintain the property of degrading the 1 ,2-DCA for a very long time, without it being necessary to add a further volume of said culture in order to guarantee the bioremediation effect.

However, the degradation speed of the pollutant could be too low with respect to the desired one.

To speed up the bioremediation process of the body of water, in a preferred aspect of the invention, the method described above could comprise a further step c):

c) feeding said body of water with an aqueous solution, optionally buffered to a pH comprised between 6.0 and 8.0, comprising at least one electron donor compound. As underlined above, the electron donor compound has the function of stimulating the reductive dehalogenation activity and promoting the maintenance of the anaerobic conditions.

In the method according to the present invention, any electron donor compound can be used, selected, for example, from organic acids and salts thereof, sugars, alcohols, or other carbon-based substrates, which stimulate the activity of the microorganisms and promote the establishment of anaerobiosis.

In a preferred aspect, said at least one electron donor compound is used in a form that promotes its slow release in the body of water (for example, as a compound in polymer form such as polylactate).

In a preferred aspect, said at least one electron donor compound is a salt of an alkaline metal or alkaline earth metal of an organic acid selected from the group comprising lactic acid, succinic acid, pyruvic acid, benzoic acid, propionic acid, formic acid and mixtures thereof and more preferably said electron donor compound is sodium lactate.

Preferably, said at least one electron donor compound is added to the body of water in quantities such as to obtain a final concentration in said body of water comprised between 0.1 mM and 5 mM, and preferably comprised between 1 mM and 2 mM.

Any buffer system known to a person skilled in the art may be used for optionally buffering the aqueous solution fed to said body of water in step c) of the method according to the invention. The function of the buffer system is also that of correcting any pH excesses in the water present in the body of water to be treated with the consortium according to the present invention.

Preferably, said aqueous solution is buffered with a phosphate buffer system at pH comprised between 6.5 and 8.0, more preferably at pH comprised between 7.0 and 7.5. Preferably said buffer system is present in said aqueous solution in concentrations comprised between 1 mM and 500 mM, more preferably between 10 mM and 250 mM. The aqueous solution of said electron donor compound, optionally buffered, may be treated prior to use for the purpose of removing the oxygen contained therein, for example through degassing, and/or insufflation of inert gases, such as nitrogen, helium and argon. However, it is important to note that the additional step c) of the method in accordance with the invention may also be performed by adding to the body of water said aqueous solution comprising said electron donor compound, optionally buffered, without removing the oxygen dissolved therein, without any perturbation being observed in the

dehalogenating activity performed by the consortium NCI MB 42687 placed in contact with the body of water.

It is therefore clear that, although it comprises anaerobic microorganisms, the consortium NCIMB 42687 (and in particular the species that expresses the dehalogenation enzyme activity, i.e. Geobacter sp.) can withstand temporary exposure to moderate quantities of dissolved oxygen without detrimental effects to the enzyme activity or to the survival of the microorganisms.

However, in the case in which the body of water treated is particularly rich in dissolved oxygen, it is possible to reduce the concentration of said dissolved oxygen, for example through the insufflation into said body of water of inert gases such as nitrogen, helium or argon, or through the use of antioxidant chemical agents (so called "oxygen scavengers") or through the use of cultures of obligated anaerobic microorganisms, in presence of appropriate nutrients.

The degree of anaerobiosis can be determined by measuring the concentration of dissolved oxygen and the potential redox of the body of water, through electrochemical measurements, for example with multiparameter probes, in accordance with standard methods known to a person skilled in the art, such as, ASTM D1498 - 14 and ASTM D888 - 12.

The method for the bioremediation of a body of water contaminated by 1 ,2-DCA, in accordance with the present invention, preferably performed in situ, represents an advantageous alternative to the bioremediation of said body of water by autochthonous dehalogenating microorganisms, in particular when the aforementioned autochthonous dehalogenating microorganisms are completely absent or are present at ineffective concentrations for the purpose of bioremediation, even after stimulation with appropriate electron donor compounds, as described above, or when the concentration of 1 ,2-DCA in the body of water exceeds a threshold value, preferably greater than or equal to 1000 mg/L, beyond which inhibiting or toxic effects are displayed on the aforementioned autochthonous microorganisms.

The degradation speed of the 1 ,2-DCA by the consortium NCIMB 42687 depends on the initial concentration of the contaminant and can be determined through gas chromatography analysis on the headspace of samples taken directly from the

microcosms. Preferably, the speed is comprised between 5 and 100 ppm of 1 ,2-DCA degraded/day, more preferably comprised between 5 and 200 ppm of 1 ,2-DCA degraded/day, even more preferably between 6 and 10 ppm of 1 ,2-DCA degraded/day. Example 4 describes a bioremediation test on an aquifer contaminated with 1 ,2-DCA and in which the bioremediation efficacy by the consortium NCIMB 42687 is demonstrated, after the addition of an aqueous solution of an electron donor compound, in accordance with step c) of the method according to the present invention.

The results of this experiment are shown in Figure 4.

Sampling performed at time intervals of about 30 days in a period of about 160 days on the aforementioned aquifer from which the consortium NCIMB 42687 was isolated to which an electron donor compound was supplied, have enabled the efficacy of said electron donor compound in substantially increasing the degradation speed of 1 ,2-DCA by the consortium itself to be confirmed.

On the samples taken over this period of time, an analysis of the metagenomic DNA was also performed, which confirmed the co-presence over time of the species of

microorganisms of the consortium NCIMB 42687.

Furthermore, with the aim of excluding the effect of an apparent reduction of the pollutant connected with scouring phenomena, the effective degradation of the 1 ,2-DCA by the dehalogenating activity associated with the consortium NCIMB 42687, was demonstrated by subjecting the samples taken from the aquifer to "Compound Specific Isotopic

Analysis" (CSIA): the isotopic analyses (Solid Phase Microextraction - Gas

Chromatography - Isotope Ratio Mass Spectrometry, SPM-GC-IRMS) were performed as described by M. Schmidt et al., in "Comparison of 1 ,2-dichloroethane, dichloroethene and vinyl chloride carbon stable isotope fractionation during dechlorination by two

Dehalococcoides strains" (2014), Water Research, vol. 52, pag. 146-154, using C0 ₂ as the reference gas, on a Delta V™ device made by ThermoFisher Scientific.

Said analyses highlighted a significant variation over time in the isotropic composition (5 ¹³ C (%o)), connected with an increase in the isotopic ratio ¹³ C/ ¹² C in the molecules of 1 ,2- DCA present in the samples taken from the aquifer. Since the enzymes, in particular the 1 ,2-DCA dehalogenase enzyme, have a preference for substrates containing the isotope ¹² C, evidence of the increase in the isotropic ratio ¹³ C/ ¹² C shows that the 1 ,2-DCA is effectively subject to enzyme degradation by the consortium according to the present invention.

In a preferred aspect, the aforementioned method may be used for the bioremediation of an aquifer contaminated by 1 ,2-DCA in which there are substantially no autochthonous dehalogenating microorganisms present.

As already mentioned, the method according to the present invention can be performed both in situ and on site and is preferably performed in situ. Any contaminated body of water may be treated with the consortium according to the present invention. In particular, the consortium NCIMB 42687 may be placed in contact with bodies of water, for example: ground water, natural and/or artificial basin water, waste water of industrial origin, percolating water from landfill, waste water of domestic origin, etc. contaminated by 1 ,2- DCA, preferably in concentration greater than or equal to 1000 mg/L, more preferably comprised between 1000 and 4000 mg/L, even more preferably comprised between 2000 and 4000 mg/L, in order to degrade the 1 ,2-DCA itself.

For the purpose of putting the present invention into practice and illustrating it more clearly, below are some non-limitative examples. Example 1 (Isolation of the consortium of microorganisms NCIMB 42687) From a piezometric well placed inside an area of about 250 m ² present in an industrial site and characterized by "chronic" pollution by 1 ,2-DCA, ten 1000 mL samples of water were taken, subsequently insufflated with gaseous N ₂ for 16 hours. From the aforementioned samples, 20 microcosms were prepared in an anaerobic hood (each sample represented in double), taking 98 mL of water from each 1000 mL sample, which were split into two sterile 55 mL bottles containing: 0.5 mL of "BTZ-3 100x" medium (NH ₄ CI 4.3 mg/L, KH ₂ P0 ₄ 50 mg/L, MgCI ₂ -6H ₂ 0 20 mg/L, CaCI ₃ -2H ₂ 0 1 mg/L and buffer HEPES 50 μΜ, brought to pH 7.5 with NaOH), cysteine hydrochloride (4 mg/mL), 0.4 mL/L of "Trace Metals Solution 200x" (Nitrilotriacetic acid 12.8 g/L, FeCI ₃ -6H ₂ 0 1 ,35 g/L, MnCI ₆ -4H ₂ 0 0,1 g/L, CoCI ₂ -6H ₂ 0 24 mg/L, CaCI ₂ -2H ₂ 0 0,1 g/L, ZnCI ₂ 0,1 g/L, CuCI ₂ -2H ₂ 0 25 mg/L, H ₃ B0 ₃ 10 mg/L, Na ₂ Mo0 ₄ -2H ₂ 0 24 mg/L, NaCI 1 g/L, NiCI ₂ -6H ₂ 0 0,12 g/L, Na ₂ Se0 ₃ -2H ₂ 0 26 mg/L), 50 μί of "Seven Vitamins Solution 1000x" (cobalamin 100 mg/L, p-aminobenzoic acid 80 mg/L, D(+)-biotin 20 mg/L, nicotinic acid 200 mg/L, calcium pantothenate 100 mg/L, pyridoxine-HCI 300 mg/L, thiamine-HCI-2H ₂ 0 200 mg/L), and 100 μί of an aqueous solution of 1 M sodium lactate. Prior to use, all the solutions were sterilized by filtration and made anaerobic through insufflation of N ₂ for 10 minutes. At the end, 1 mL of a sterile aqueous solution of 1 ,2-DCA 5000 mg/L was added. The total volume of liquid in the microcosm bottles therefore amounts to about 51 .05 mL.

Subsequently, the bottles, maintained under anaerobic conditions, were closed with a Teflon ^® cap and sealed with an aluminum ring. The microcosms were kept at 26°C without agitation and in the dark.

After 7 days a 1 mL sample was taken from each microcosm on which headspace gas chromatography analysis was performed. For the analysis, 1 mL of culture of each microcosm was placed in a 20 mL glass vial containing 9 mL of distilled water and 4 g of NaCI. Each sample was then incubated at 80°C for 20 minutes under moderate agitation. After that 1 μΙ_ taken from the headspace was injected into gas chromatography equipped with a DB-5MS Ul column (Agilent Technologies, 60 m, 0.25 mm ID, 0.25 μηη film).

All the cultures highlighted comparable degradation activity on the 1 ,2-DCA present in the medium. One of these was subjected to some refreshment steps with sterile medium prepared as described above and a tolerance test to the 1 ,2-DCA was performed on the microcosm obtained.

To do this, from the microcosm in question, five microcosms in duplicate were prepared through refreshment, as described above with the difference that the 1 ,2-DCA was added in the five microcosms at respective final concentrations of 50 mg/L, 300 mg/L, 1000 mg/L, 2000 mg/L and 4000 mg/L. Also in this case, the microcosms were kept at 26°C without agitation and in the dark. On pre-fixed days, a sample was taken from each microcosm on which headspace gas chromatography analysis was performed as described above.

Figure 3 shows a graph of the percentage variations corresponding to the reduction in concentration of 1 ,2-DCA (Figure 3a) and the increase in concentration of ethylene

(Figure 3b) measured after 3, 14 and 17 days of growth of the culture in the different microcosms characterized by increasing concentrations of 1 ,2-DCA.

The consortium of microorganisms of the aforementioned culture was deposited at the

"National Collection of Industrial Food and Marine Bacteria" (NCIMB) in Aberdeen,

Scotland, UK, and the filing number NCIMB 42687 was assigned thereto.

Example 2 (Sequencing of the metagenome of the consortium of microorganisms NCIMB 42687 and identification of the 16S rDNA)

The isolation of the genomic DNA of the microorganisms that constitute the consortium NCI MB 42687 was performed with the kit "FastDNA® SPIN Kit for Soil" (MP Biomedicals) following the instructions contained in the protocol "Ion Torrent Plus gDNA Fragment library preparation User guide 4471989 revD" of the manufacturer Life Technologies. In particular, the DNA was fragmented enzymatically with the "Ion Shear Plus Reagents Kit" used in accordance with the instructions provided by the manufacturer. The DNA thus treated was bound to the P1 adapters and bar-coded adapter 2 in accordance with the protocol mentioned above and therefore subjected to agarose gel electrophoresis. The fragments with an average length around 300-400 bp were eluted by the agarose gel and purified. The DNA of three "bar coded libraries" of fragments obtained as described above was quantified using the BioAnalyzer tool according to the manufacturer's protocol.

Thereafter the bar coded libraries were mixed following the protocol itself and subjected to sequencing on Ion 316 chip (v2) following the instructions contained in the "lonTorrent Ion PGM Sequencing 400 kit" manual (Publ number MAN0007242).

The sequences of DNA obtained were compared with sequences of DNA contained in the non-redundant "Bacteria" database using the software BLASTN® (NCBI, U.S. National Library of Medicine) and aligned with sequences of 16S bacterial ribosomal DNA through the program "Local Blast" (BioEdit) using the file of total sequences (over 1 ,8- 10 ⁶ bases) as the "Local Nucleotide Database File".

In this way it was possible to identify four different sequences of 16S rDNA, corresponding to the four SEQ ID NO sequences: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, which are homologous to sequences corresponding to the preponderant species of microorganisms of the anaerobic consortium NCIMB 42687, i.e. Geobacter sp., Pseudomonas sp., Clostridium sp. and Acidovorax sp.

In particular, in the set of sequences corresponding to the composition of the consortium from whose culture the metagenomic DNA analyzed was extracted, a quarter of the total sequences with a coverage index equal to 6,2x can be attributed to rDNA 16 S sequences of Geobacter sp.

Example 3 (Identification of the genomic DNA which comprises the genes correlated to 1 ,2-dichloroethane dehalogenases of Geobacter sp.).

Starting from the file of total sequences constituting the "Local Nucleotide Database File", obtained as described in Example 2 above, and using as a reference the sequences of genes correlated with the dehalogenase of Desulfitobacterium dichloroeliminans

(described by M. Marzorati, et al. in "A novel reductive dehalogenase, identified in a contaminated groundwater enrichment culture and in Desulfitobacterium dichloroeliminans strain DCA1 , is linked to dehalogenation of 1 ,2-dichloroethane" (2007) Applied and Environ. Microbiol., vol. 73, pag. 2990-2999), through the program "Local Blast" (BioEdit) nucleotide sequences have been identified corresponding to the genes rdhA, rdhB, rdhC and rdhT in a region of DNA of about 4000 pairs of bases, which is flanked by

characteristic sequences of Geobacter sp.

The aforementioned region of DNA is represented schematically in Figure 1 . The sequences of the four genes are identified with the SEQ ID NO codes: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

Example 4 (Bioremediation test in situ in an aquifer with the consortium NCI MB 42687) For the purpose of verifying the efficacy of the consortium NCI MB 42687 in situ, the variation in the concentration of 1 ,2-DCA in water contained in the piezometric well mentioned in Example 1 was measured, i.e. in the same aquifer from which the consortium was isolated, after stimulation of the dehalogenation activity of said consortium with an electron donor compound.

For that purpose, 7000 L of an aqueous solution of sodium lactate were prepared, obtained by diluting in water 450 kg of a commercial solution of 60% sodium lactate by weight in water.

The aqueous solution of sodium lactate was equally split and pumped into four injection wells especially created in proximity to the aforementioned piezometric well and comprised in the 250 m ² area already described in Example 1 .

At prefixed intervals of time, a sample of water was taken from the aforementioned piezometric well, that was analyzed to measure the variation in concentration of 1 ,2-DCA through gas chromatography, as described in Example 1.

The results of the gas chromatography analysis are reported in Table 1 and presented in Figure 4.

As per the results, the concentration of 1 ,2-DCA in solution in the water in the piezometric well in which the consortium NCIMB 42687 is present stimulated with sodium lactate as the electron donor passes from over 4000 mg/L to 122 mg/L (variation: -97%).

Table 1 : variation in the concentration of 1 ,2-DCA in solution

The average degradation speed is seen to be about 10 times higher than the degradation speed observed prior to the treatment.

Starting from the samples taken, microcosms were prepared and maintained from which the metagenomic DNA was extracted and sequenced. In all the samples it was possible to confirm the persistence of the consortium NCIMB 42687.

Finally, it is however to be understood that further changes and variations may be made to the process described and illustrated herein which do not depart from the scope of the appended claims.