Method for consolidating substrates by cathodic polarization and biocalcifying bacteria

Abstract

The present invention relates to the field of environmental engineering and, more particularly, to a method for consolidating substrates by cathodic polarization and biocalcifying bacteria. The present invention relates to an in-situ method for consolidating a substrate, the method comprising the steps of: i) inoculating a medium comprising the substrate with biocalcifying bacteria; ii) applying cathodic polarization with an imposed current density. The invention also relates to an ex-situ method for consolidating a substrate, the method comprising the steps of: i) mixing the biocalcifying bacteria with a substrate; ii) adding a cementation solution to the mixture obtained in step i); iii) applying cathodic polarization with an imposed current density.

Description

Title: Process for Consolidating Substrates by Cathodic Polarization and Biocalcifying Bacteria technical field

[0001] The present invention falls within the field of environmental engineering, and relates more particularly to a process for consolidating substrates by cathodic polarization and biocalcifying bacteria. Previous technique

[0002] Coastal erosion poses a serious threat as it causes the degradation of dunes, beaches, cliffs, and the deterioration of seawalls. Coastal erosion is defined as the permanent loss of land along the coastline, resulting in the transformation of the coast. Over the past 50 years, nearly 20% of France's natural coastline has receded, and approximately 30 km 2land has disappeared. In order to protect coastal areas and structures, it is necessary to find an effective way to combat erosion with a low environmental impact (Vincent, J.; Sabot, R.; Lanneluc, I.; Refait, P.; Turcry, P.; Mahieux, P.-Y.; Jeannin, M.; Sablé, S. Biomineralization of Calcium Carbonate by Marine Bacterial Strains Isolated from Calcareous Deposits. Matériaux & Techniques 2020, 108, 302, doi: 10.1051 / mattech / 2020027).

[0003] Biocalcification refers to the biological process of biomineralization by which certain microorganisms induce the precipitation of calcium carbonate (CaCO₃) in their environment. So-called biocalcifying bacteria create conditions favorable to calcium carbonate precipitation through certain metabolic activities, such as the production of carbonates by carbonic anhydrase and the alkalinization of the environment by urease.

[0004] Applications in the field of biocalcification include the stabilization of terrestrial, coastal, and marine environments, the strengthening of construction materials, and carbon dioxide (CO2) sequestration. In coastal and marine applications, biocalcification can help protect coastlines from erosion, stabilize marine infrastructure, and consolidate sediments on beaches and seabeds. In construction, biocalcification can be used to manufacture biomaterials and can improve mechanical properties, repair cracks, and increase the durability of existing structures. Furthermore, biocalcifying bacteria can contribute to environmental sustainability by capturing and storing CO2 as calcium carbonate.

[0005] The challenges in achieving these application goals include the need for efficient and environmentally friendly methods to induce biocalcification. Traditional methods often rely on urease-producing bacteria, which require the addition of urea. This process produces ammonia, a toxic byproduct that can harm the environment. Furthermore, the use of exogenous bacteria can disrupt local ecosystems and biodiversity. Therefore, it is necessary to develop methods that utilize endogenous bacteria and avoid the production of harmful byproducts.

[0006] It is known from prior art that the company Soletanche Bachy developed a biocementation technology called Biocalcis®. This technology involves the pressureless injection of a bacterial suspension and cementation solution. The process uses the bacterial strain Sporosarcina pasteurii, which requires the use of urea to induce the precipitation of calcium carbonate. However, the production of ammonia as a byproduct limits the use of this method in natural environments, particularly marine environments.

[0007] It is known from prior art that cathodic polarization is a process that induces the abiotic precipitation of calcareous and magnesian compounds on a metallic surface through electrochemistry. This method protects metallic structures against corrosion and can be used to stabilize coastal areas by forming a calcareous-magnesian deposit that binds sediments. However, the formation of these deposits takes a long time, often several years, to reach a significant thickness. The process is also limited to areas that are frequently or completely submerged. Furthermore, cathodic polarization can lead to a substantial increase in the pH of the environment and inhibit the growth of endogenous microorganisms. Exposed

[0008] The present invention relates to a method for consolidating substrates using a combination of cathodic polarization and biocalcifying bacteria.

[0009] Developing the processes according to the invention required overcoming numerous major technical uncertainties that called its feasibility into question. Indeed, several critical factors raised legitimate doubts: the potential inhibition of bacterial growth due to increased pH at high polarizations, the uncertain ability of bacteria to adhere permanently to the granular matrix upon application of the cementing solution, the ability of bacteria to withstand cathodic polarization in a granular matrix, and their capacity to survive and grow in this challenging environment. Furthermore, the ability of bacteria to proliferate in anoxic zones near the positive electrode and their potential for growth in seawater without added nutrients were major concerns.

[0010] The inventors were able to provide technical solutions to overcome all of these difficulties, and experimental results demonstrated that these different constraints could be controlled.

[0011] Indeed, it has been demonstrated that the process according to the invention makes it possible to generate a high-performance limestone network, giving superior mechanical properties to an initially non-cohesive substrate.

[0012] This innovation opens up new perspectives for environmental geotechnical stabilization, particularly in coastal protection, strengthening of marine structures, repair of damaged materials and design of biomaterials.

[0013] Furthermore, experimental work demonstrates the superiority of the process, producing a calcomagnesian agglomerate significantly thicker than conventional methods using only cathodic polarization for the same treatment time.

[0014] Another major advantage lies in its compatibility with natural seawater, eliminating the need for complex chemical solutions and reducing operational costs.

[0015] This innovative process is also distinguished by its environmental excellence: the use of indigenous bacteria, low CO2 emissions, and the absence of urea eliminate the risk of toxic ammonia emissions. Cathodic polarization induces alkalinization, maximizing the efficiency of bacterial biocalcification and CaCO3 formation. Figures

[0016] Figure 1: Experimental column biocementation setup for the formation of a sandy-calcareous agglomerate by biocalcifying bacteria

[0017] Figure 2: Syringe biocementation setup for the formation of a sandy-calcareous agglomerate by biocalcifying bacteria associated with cathodic polarization. The setup is fed with a cementation solution from bottom to top by a peristaltic pump (discontinuous or continuous flow), and connected to a potentiostat that allows a current to be imposed between a platinum anode and a carbon steel cathode buried in the sand in the syringe.

[0018] Figure 3: Calcomagnesian agglomerates obtained without (A) and with 10-fold concentrated and lyophilized bacteria (B) after 1 month of treatment with continuous circulation in a cementation solution (flow rate = 0.03 mL.min-1) composed of natural seawater sterilized by filtration, enriched only with 25 mmol / L CaCh, with an imposed current density of -150 pA.cm 2 based on a sequential polarization cycle (every 6 hours).

[0019] Figure 4: Column biocementation setup for the formation of a sandy-calcareous agglomerate by biocalcifying bacteria associated with cathodic polarization. The setup is fed with a cementation solution from bottom to top by a peristaltic pump (discontinuous or continuous flow), and connected to a potentiostat which allows a current to be imposed between a platinum anode and a carbon steel cathode, buried in the sand in the syringe.

[0020] Figure 5: E5 trial before and after one month of treatment

[0021] Figure 6. Evolution of bacterial growth, pH, and bioprecipitated CaCO₃ concentration by the biocalcifying bacterial strains CD4 and MD1 in different media: MB-CaCh, MB-Ac.Ca, C-CaCh physic water, CaCh physic water, and Ca-Ac.Ca physic water

[0022] The present invention covers an in-situ substrate consolidation process comprising the steps of: i) Seeding of a medium comprising the substrate with biocalcifying bacteria ii) Application of cathodic polarization, preferably cathodic polarization with an imposed current density.

[0023] The present invention also covers an ex situ substrate consolidation process comprising the steps of: i) Mixing of biocalcifying bacteria with a substrate, ii) Addition of a cementation solution to the mixture obtained in step i) iii) Application of cathodic polarization, preferably cathodic polarization with an imposed current density.

[0024] Advantageously, the processes according to the invention make it possible to create a resistant intergranular limestone network which improves the mechanical properties of the substrate, thus allowing its use for applications such as coastal protection and stabilization of marine structures (in situ process) as well as the repair of materials (ex situ process).

[0025] Advantageously, cathodic polarization allows the medium to be alkalinized and promotes the precipitation of CaCO₃ by biocalcifying bacteria. It eliminates the need for alkalinization caused by the hydrolysis of urea by ureolytic bacteria.

[0026] in situ process

[0027] The term "in situ process" refers to a consolidation process carried out directly in the natural or usual environment of the substrate to be consolidated, without moving it.

[0028] For the purposes of the present invention, "medium" means the specific environment in which the consolidation process is implemented, i.e., the medium comprising the substrate and in which the biocalcifying bacteria will be seeded to induce the bioprecipitation of calcium carbonate (CaCOs).

[0029] For illustrative purposes, the environment can be the sea, oceans, coastline, marine structures, seabed, soils, banks, slopes, embankments, limestone rocks, dikes, foundations, retention basins, bridge piers, concrete-based infrastructure, port structures.

[0030] As an illustration, the in situ consolidation process will be used for environmental geotechnical stabilization.

[0031] Ex situ process

[0032] The term "ex situ process" refers to a consolidation process carried out in an environment different from the natural or usual environment of the element to be consolidated, requiring the movement of the latter.

[0033] As an illustration, the ex situ consolidation process will be used for the rehabilitation or design of materials.

[0034] Biocalcifying bacteria

[0035] Biocalcifying bacteria are microorganisms capable of creating local conditions favorable to the precipitation of calcium carbonate (CaCO₃) by supplying carbonate ions or by alkalizing the environment into which they are introduced. This biomineralization process, called biocalcification, occurs through several bacterial enzymatic activities that can act synergistically, such as carbonic anhydrase and urease.

[0036] According to one embodiment, biocalcifying bacteria can be ureolytic.

[0037] The term “ureolytic biocalcifying bacteria” refers to bacteria capable of inducing the precipitation of CaCCh in the presence of urea and involving the hydrolysis of urea by a urease.

[0038] According to one embodiment, biocalcifying bacteria can be non-ureolytic.

[0039] The term “non-ureolytic biocalcifying bacteria” refers to bacteria capable of inducing the precipitation of CaCCh in the absence of urea.

[0040] Indeed, these bacteria possess other metabolic pathways responsible for biocalcification such as the formation of carbonate ions by carbonic anhydrase, denitrification, ammonification of amino acids, oxidation of organic acids, oxidation of methane, or reduction of sulfates.

[0041] Carbonic anhydrase is a key enzyme that catalyzes the interconversion of CO2 to bicarbonate (HCO3). Under alkaline conditions, the carbonates produced by carbonic anhydrase precipitate as CaCCh provided there is a sufficient concentration of Ca 2+ in the environment.

[0042] Denitrification is the reduction of nitrates (NO3) to nitrogen gas (N2) and is carried out by denitrifying bacteria. Nitrate reduction occurs during the microbial oxidation of organic matter in the absence of oxygen. This process induces alkalinization and an increase in carbonate concentration, thus promoting the precipitation of CaCO3 (Er§an, Y.C. et al 2016 https: / / doi.Org / 10.1016 / j.cemconres.2016.01.009). Their action in the absence of oxygen could be utilized in close proximity to polarized steel in an anoxic environment.

[0043] Ammonification is the process by which microorganisms produce ammonium (NH4). +) by mineralizing dissolved organic molecules of low molecular weight, containing amine or amide groups such as amino acids, urea, amino sugars, and nucleotides (Romillac, 2019 = Romillac, N. (2019). Ammonification. In Encyclopedia of Ecology (Second Edition), B. Fath, ed. (Oxford: Elsevier), pp. 256-263). The ammonification of amino acids produces, in particular, ammonium and hydroxide ions around the cell, consequently promoting the precipitation of CaCO₃ (Zhu and Dittrich, 2016 https: / / doi.orq / 10.3389 / fbioe.2016.00004).

[0044] The oxidation of certain organic acids, such as acetate, lactate, citrate, succinate, oxalate, malate, and glyoxylate, allows the release of sufficient CO2, produced by cellular respiration, to increase the concentration of carbonate ions (Riding and Awramik (eds) 2000, Microbial sediments, Springer). Indeed, the CO2 released by the cell reacts with water molecules to produce bicarbonate (HCO3) and carbonate (CO3) ions. 2 ) under alkaline conditions. This increase in carbonate ion concentration thus promotes the precipitation of CaCCh if the medium contains Ca2 + .

[0045] The oxidation of methane also produces CO2, which is released outside the cell and, in the presence of water, converts to HCO3-, which can precipitate as CaCCh provided there is a sufficient quantity of Ca2+. +free. This metabolic pathway has been identified in methanotrophic bacteria that can induce CaCO3 precipitation (Ganendra et al., 2014 https: / / doi.org / 10.1128 / AEM.01349-14).

[0046] Under anaerobic conditions, certain bacteria, such as sulfate-reducing bacteria (SRB), favorably modify pH, carbonate concentration, and / or calcium activity, while simultaneously reducing sulfates to sulfides (Deng et al., 2010 https: / / doi.org / 10.1016 / j.chemgeo.2010.09.008). The complete oxidation of organic matter releases CO2, which, upon contact with water, forms carbonic acid (H2CO3). The reduction of sulfates to sulfides induces a sufficient increase in pH to transform carbonic acid into bicarbonate (HCO3) and carbonate (CO3) ions. 2 ) promoting the precipitation of CaCO3 (Paul et al., 2017 https: / / doi.Org / 10.1016 / j.apgeochem.2017.01.010).

[0047] A professional skilled in the art can recognize biocalcifying bacteria by their ability to induce the precipitation of calcareous mineral phases. To characterize this property, they can employ all the analytical techniques known in the field. In particular, Raman microspectrometry, thermogravimetric analysis, and X-ray diffraction are preferred methods for identifying and characterizing the mineral deposits formed. These techniques allow for the unambiguous confirmation of the presence of calcium and / or magnesium-based mineral phases, the formation of which is induced by the metabolic activity of biocalcifying bacteria and by cathodic polarization.

[0048] The metabolic pathways of biocalcifying bacteria can be characterized by specific enzymatic assays. Those skilled in the art can implement various enzymatic characterization methods known in the art. In particular, urease activity is demonstrated using a urea-indole medium, where the degradation of urea by the enzyme induces a color change characteristic of the medium. Carbonic anhydrase activity, on the other hand, is demonstrated using a substrate, para-nitrophenyl acetate, which is transformed into para-nitrophenol, a yellow compound, by the action of this enzyme. Those skilled in the art can confirm carbonic anhydrase activity using inhibitors specific to this enzyme or by amplifying the genes encoding this enzyme by PCR with specific primers.

[0049] Identifying these bacteria by sequencing the complete bacterial genome or by sequencing 16s rDNA can confirm the results obtained by enzymatic tests by providing additional information on their metabolic potential.

[0050] For illustrative purposes, the essentials of these methods are described in Vincent, J.; Sabot, R.; Lanneluc, I.; Refait, P.; Turcry, P.; Mahieux, P.-Y.; Jeannin, M.; Sablé, S. Biomineralization of Calcium Carbonate by Marine Bacterial Strains Isolated from Calcareous Deposits. Matériaux & Techniques 2020, 108, 302, doi:10.1051 / mattech / 2020027, and Vincent, J.; Colin, B.; Lanneluc, I.; Sabot, R.; Sopéna, V.; Turcry, P.; Mahieux, P.-Y.; Refait, P.; Jeannin, M.; Sablé, S. New Biocalcifying Marine Bacterial Strains Isolated from Calcareous Deposits and Immediate Surroundings. Microorganisms 2022, 10, 76, doi:10.3390 / microorganisms10010076.

[0051] According to one embodiment, non-ureolytic biocalcifying bacteria induce the precipitation of calcium carbonate (CaCOs) via carbonic anhydrase.

[0052] According to this embodiment, the bacteria induce the precipitation of calcium carbonate (CaCOs) in the absence of urea, which reduces the environmental impact and avoids the production of toxic ammonia.

[0053] According to one embodiment, the bacteria may be chosen from among gammaproteobacteria, alphaproteobacteria, firmicutes and mixtures thereof.

[0054] Typically, gammaproteobacteria can be chosen from Pseudoalteromonas sp. and Pseudidiomarina maritima, alphaproteobacteria can be chosen from Epibacterium mobile and firmicutes can be chosen from Virgibacillus halodenitrificans, Pianococcus maritimus, Bhargavaea beijingensis, and Bhargavaea ginsengi.

[0055] According to one embodiment, the bacteria are selected from: - MD1 Pseudoalteromonas sp.. - SW1 Pseudoalteromonas sp. - SW2 Pseudoalteromonas sp. - SW3 Pseudoalteromonas sp - CD9 Pseudoalteromonas sp. - CD 10 Pseudoalteromonas sp. - CD3 Pseudidiomarina maritima - CD1 Epibacterium motile - CD4 Epibacterium motile - CD5 Epibacterium motile - CD6 Virgibacillus halodenitrificans - CD8 Pianococcus maritimus - CD2 Bhargavaea ginsengi, - CD7 Bhargavaea beijingensis and their mixtures.

[0056] According to one embodiment, the bacteria are chosen from CD4 Epibacterium mobile, MD1 Pseudoalteromonas sp., CD8 Pianococcus maritimus and their mixtures.

[0057] According to a preferred embodiment, the bacteria are selected from CD4 Epibacterium motile, MD1 Pseudoalteromonas sp. and their mixtures.

[0058] The 16S rDNA gene sequences of the 14 strains have been deposited in the GenBank database. The accession numbers are MW774399 to MW774412:

[0059] [Table 1] Bacteria Genbank Accession Numbers MD1 Pseudoalteromonas sp. MW774412 SW1 Pseudoalteromonas sp. MW774409 SW2 Pseudoalteromonas sp. MW774410 SW3 Pseudoalteromonas sp. MW774411 CD9 Pseudoalteromonas sp. MW774407 CD 10 Pseudoalteromonas sp. MW774408 CD3 Pseudidiomarina maritima MW774401 CD1 Epibacterium mobile MW774399 CD4 Epibacterium mobile MW774402 CD5 Epibacterium mobile MW774403 CD6 Virgibacillus halodenitrificans MW774404 CD8 Pianococcus maritimus MW774406 CD2 Bhargavaea ginsengi MW774400 CD7 Bhargavaea beijingensis MW774405

[0060] According to one embodiment, biocalcifying bacteria are used as such or in concentrated and / or lyophilized form.

[0061] In one embodiment, biocalcifying bacteria are incorporated into the substrate either whole or in concentrated form. The substrate and incorporated bacteria (whole or in concentrated form) are then lyophilized to obtain a lyophilized substrate containing the biocalcifying bacteria. Bacterial concentration and lyophilization are common techniques well understood by those skilled in the art. Concentration can be achieved using conventional methods such as centrifugation or filtration, while lyophilization involves freezing followed by dehydration under vacuum, resulting in a stable and shelf-stable form of the bacteria.

[0062] As an illustration, the concentration is achieved by centrifugation (5000g for 5 min) of a bacterial suspension that has grown for 24 hours. The supernatant is then discarded, and the pellet is resuspended in physiological saline (distilled water + 9 g / L NaCl) in a volume sufficient to concentrate the bacterial concentration tenfold.

[0063] In one embodiment, the biocalcifying bacteria are used in concentrated form. The bacterial suspension used may be a concentrated suspension relative to a reference suspension, in particular at a concentration of 2X. In some embodiments, the concentration of the bacterial suspension is 3X, 4X, 5X, 6X, 7X, 8X, 9X, or 10X. In a preferred embodiment, the bacterial suspension is a 10X concentrated suspension.

[0064] The bacterial suspension (with or without concentration step) is frozen at -80 °C and lyophilized by a drying process at a pressure below 450 mTorr at -80 °C (COSMOS 20K lyophilizer) for 4 days.

[0065] Inoculating lyophilized and / or concentrated bacteria into the medium (in situ procedure) and mixing them with the substrate (ex situ procedure) are common microbiological operations. Those skilled in the art are familiar with the appropriate techniques for performing these operations, whether they involve inoculation methods in liquid or solid media, or mixing processes that ensure homogeneous distribution of bacteria within the substrate.

[0066] In a preferred embodiment, biocalcifying bacteria are incorporated into the substrate, either whole or in concentrated form, and then the bacteria-substrate mixture is freeze-dried. This method yields a ready-to-use material that is easier to store and handle, and directly incorporates the elements necessary for carrying out the process.

[0067] The following embodiments are described in relation to the in situ process.

[0068] According to one embodiment, the process includes a preliminary step of selecting biocalcifying bacteria from the medium.

[0069] The selection step may include any method known to a person skilled in the art, such as performing at least one of the following tests or a combination thereof: enzymatic tests, validation of the presence of CaCO3 induced by bacteria, complete sequencing of the bacterial genome, or sequencing of their 16S rDNA.

[0070] Typically, the selection stage may include at least one of the following tests or a combination thereof: - characterization of urease activity using a urea-indole medium, - the characterization of carbonic anhydrase activity using para-nitrophenyl acetate as a substrate, - validation of the presence of CaCO₃ crystals induced by bacteria, by Raman microspectrometry - sequencing the complete genome of bacteria after extraction of genomic DNA, - Sequencing of 16s rDNA from bacteria after extraction of genomic DNA and PCR amplification of the 16S rDNA gene.

[0071] For carbonic anhydrase activity, a person skilled in the art can confirm carbonic anhydrase activity by using inhibitors specific to this enzyme or by amplification of the genes encoding this enzyme by PCR with specific primers.

[0072] The person skilled in the art knows how to implement these different characterization methods which are known in the art and widely documented in the scientific literature.

[0073] According to one embodiment, biocalcifying bacteria are endogenous bacteria of the environment.

[0074] By "endogenous bacteria" we mean bacteria naturally present in the environment in question, that is to say bacteria which originate and have naturally developed in this environment, as opposed to bacteria which would be artificially introduced or which would come from another environment.

[0075] The use of native bacteria offers several advantages. These bacteria are already adapted to the specific physicochemical conditions of the environment. Furthermore, being naturally present in the environment, they are in equilibrium with the existing ecosystem and exhibit a greater capacity for survival and growth. This natural adaptation optimizes the efficiency of the process while limiting the risk of introducing external species that could disrupt the environmental balance.

[0076] Cathode polarization

[0077] Cathodic polarization is a process that induces the abiotic precipitation of calcareous and magnesian compounds (calcium-magnesian deposition) on a metallic surface by electrochemistry. The process is based on the cathodic polarization by impressed current or impressed potential of a metallic structure (D; Petrocokino, Materials maintained in the state of anode or cathode throughout use cathodic protection - cathodic protection, Technique de l'ingénieur - Ref M156 V1; D. Petocokino, Anodic protection - cathodic protection, Technique de l'ingénieur - Ref COR1010 V1; J. Creus, R. Sabot, Ph. Refait, Corrosion and protection of metals in marine environments, Techniques de l'ingénieur - ref: COR620 V2), allowing the formation of the calcareous magnesium deposit ((Carré et al. Electrochemical calcareous deposition in seawater. A review, Env. Chem. Letters, Vol 18 (2020) DOI: 10.1007 / s10311-020-01002-z), in volume around the cathode and thus cement the existing substrate (Carré et al., Electrochemical limestone synthesis in seawater binds metal grids and sediments for coastal protection, Env. Chem. Letters, vol. 18 (2020) DOI: 10.1007 / s10311-020-01019-4). The calcomagnesian deposit therefore serves as a natural binder. In one embodiment, the cathode is a structure allowing the passage of current, such as a metal grid (any type of steel) or a geotextile containing metal wires or conductive carbon fibers, or a metal wire or iron filings.

[0078] According to one embodiment, cathodic polarization was carried out on a conductive substrate, preferably metallic, which could preferably be carbon steel or galvanized steel.

[0079] For an in situ process, it will either be buried or deployed on the surface. For an ex situ process, it will be introduced into the environment according to the desired shape of the objects to be created.

[0080] According to one embodiment (in situ or ex situ process), cathodic polarization is applied to a continuous or sequential polarization cycle.

[0081] According to a particularly preferred embodiment, cathodic polarization is applied over a continuous polarization cycle.

[0082] According to one embodiment (in situ or ex situ process), the polarization is applied on a sequential polarization cycle, with a frequency between 0.01 and 23.99 hours, preferably between 4 and 8 hours, and preferably in which the cathode polarization is applied on a sequential polarization cycle every 6 hours.

[0083] According to a preferred embodiment, cathodic polarization is cathodic polarization with an imposed current density.

[0084] Thus, the present invention covers an in-situ substrate consolidation process comprising the steps of: i) Seeding of a medium comprising the substrate with biocalcifying bacteria ii) Application of cathodic polarization with an imposed current density.

[0085] The present invention also covers an ex situ substrate consolidation process comprising the steps of: i) Mixing of biocalcifying bacteria with a substrate, ii) Addition of a cementing solution to the mixture obtained in step i) iii) Application of cathodic polarization with an imposed current density.

[0086] Advantageously, cathodic polarization with imposed current density allows control of the kinetics of electrochemical reactions occurring at the metal surface. In this case, the cathode potential will adapt according to the resistance encountered to maintain a constant current.

[0087] Cathodic polarization with an imposed current density is contrasted with polarization by imposed potential, where the current flowing through the medium adapts according to the resistance of the medium and the interface to maintain a constant potential setpoint (according to Ohm's law U=RI, where U is the anode-cathode potential difference in volts, R is the resistance of the medium in ohms, and I is the current in amperes). Thus, as one of the electrodes (cathode) becomes coated with a calcareous or calc-magnesian deposit, the current will decrease and the alkalinization process will slow considerably. The deposit will stop growing. Furthermore, on the other electrode (anode), the medium will acidify and the calcareous deposit will dissolve. Such a process by imposed potential polarization is described, for example, in GUANGHUI SHAO et al. (“Application of electric fields to improve cementation homogeneity of biogrouted sands”, SOIL USE AND MANAGEMENT, WILEY-BLACKWELL PUBLISHING, GB, vol.40, no.1, July 19, 2023 (2023-07-19), page n / a, XP072609621), in which the electric field is used to make biocalcifying bacteria migrate and not to participate in the production of limestone via an alkalization of the medium, contrary to the present invention.

[0088] Thus, unlike polarization by imposed potential, the alkalinization of the electrolyte will continue to increase over time, promoting the volume growth of the calcomagnesian deposit under cathodic polarization with an imposed current density according to the invention.

[0089] According to one embodiment (in situ or ex situ process), the polarization is applied with a current density between -10 pA.cnrr 2 and -600 pA.cirr 2 , preferably between -125 pA.cirr 2 and -175 pA.cirr 2 , and preferably with a current density of -150 pA.cm 2 .

[0090] Cementing solution

[0091] The inventors have advantageously highlighted that the composition of natural seawater is sufficient to induce the precipitation of CaCO3 in the process according to the invention.

[0092] in situ process

[0093] The following embodiments are described in relation to the in situ process.

[0094] In a marine environment, that is, an environment where seawater is present, the cementation solution may consist entirely or partially of seawater. Thus, according to this embodiment, the environment is the cementation solution.

[0095] Thus, according to one embodiment, when the environment is a marine environment, the cementation solution is composed of seawater.

[0096] According to one embodiment, seawater may be enriched with nutrient broth, peptones, sugars and mixtures thereof.

[0097] According to a preferred embodiment, seawater is enriched with calcium.

[0098] According to a preferred embodiment, seawater may be enriched with calcium 25 mmol / L.

[0099] In an in situ process in a non-marine environment, that is, an environment where seawater is not present, a cementing solution is added to the medium. Thus, according to another embodiment, when the environment is not marine, a cementing solution is added to the medium.

[0100] The term “cementation solution” refers to a liquid used to induce the bioprecipitation of calcium carbonate (CaCOs) in order to consolidate a substrate.

[0101] Ex situ process

[0102] In the ex situ process, a cementation solution is mixed with the substrate.

[0103] Similarly, a "cementation solution" is a liquid used to induce the bioprecipitation of calcium carbonate (CaCOs) in order to consolidate a substrate.

[0104] In situ and ex situ process

[0105] The following paragraphs are described in relation to the ex situ process and in situ processes using a cementation solution.

[0106] According to one embodiment, the cementation solution comprises seawater and / or physiological water.

[0107] According to a preferred embodiment, the cementation solution includes seawater.

[0108] "Physiological water" refers to an aqueous solution of sodium chloride (NaCl at 9 gL 1 ).

[0109] According to one embodiment, the solution further comprises at least one nutrient selected from calcium, magnesium, organic compounds such as glucose, CO2, nitrogenous compounds such as urea, ammonium salts, nitrates, nitrites, amino acids, peptides and proteins, phosphate, potassium and mixtures thereof.

[0110] According to one embodiment, the cementation solution comprises at least one source of calcium, preferably chosen from calcium chloride and calcium acetate.

[0111] According to one embodiment, the cementation solution does not contain exogenous urea.

[0112] By "exogenous urea" we mean urea that is artificially added to the environment, that is, urea that is not naturally present in the environment or that is not produced by microorganisms present in the environment, as opposed to urea that would be naturally produced by microorganisms or naturally present in the environment.

[0113] According to one embodiment, the cementation solution comprises calcium-enriched seawater, preferably enriched with 25 mmol / L of calcium. -1 .

[0114] In some embodiments, calcium enrichment is achieved using at least one source of calcium, preferably chosen from calcium chloride and calcium acetate.

[0115] According to one embodiment, the cementation solution consists of seawater enriched with calcium, preferably enriched with 25 mmol / L of calcium. -1said seawater being enriched by means of at least one source of calcium, preferably chosen from calcium chloride and calcium acetate.

[0116] In another embodiment, the medium is not marine, so the cementation solution is not, or does not contain, seawater. In some embodiments, the cementation solution is physiological saline. In such an embodiment, the calcium source is preferably calcium acetate.

[0117] The following embodiments are described in relation to the in situ process and the ex situ process.

[0118] In one embodiment, the cementation solution is injected, in a continuous or discontinuous flow, into the medium, as part of an in situ process, or into the mixture composed of biocalcifying bacteria and the substrate, at a flow rate of between 0.001 and 1000 mL / min -1 , preferably 0.01 and 0.1 mL.min-1 , preferably between 0.02 and 0.05 mL.min -1 and preferably at approximately 0.03 mL / min -1 .

[0119] Substrate

[0120] The term "substrate" refers to any material, natural or artificial, that can be brought into contact with biocalcifying bacteria, an electric current, and, where applicable, the cementation solution, within the framework of the described process. The substrate may be in powder form, cohesive form, or as a mixture of materials of different natures.

[0121] According to one embodiment, the substrate is chosen from a powdery substrate, a cohesive substrate or a mixture of a powdery substrate and a cohesive substrate.

[0122] A "powdery substrate" is defined as a material consisting of distinct and individual particles, loosely or not at all bound together, in the form of powder or grains. These substrates include, but are not limited to, sands, gravels, limestone, mining tailings, ash, and inert industrial waste.

[0123] A "cohesive substrate" is defined as a material whose particles are bound together by attractive forces, giving it natural cohesion and a certain degree of mechanical strength. These substrates include, but are not limited to, clays, silts, mud, sediments, industrial sludge, and peat.

[0124] According to one embodiment, the substrate is chosen from sand, mud, sediments, glass beads, clay, gravel, limestone, industrial sludge, mining residues, ash, silt, peat and inert industrial waste.

[0125] Environmental Qeotechnical Stabilization

[0126] Environmental geotechnical stabilization means any application of the process aimed at strengthening, consolidating or stabilizing natural or artificial formations in their environment, for the purpose of protection, preservation or restoration.

[0127] For example, environmental geotechnical stabilization includes: - Coastal and port protection to combat coastal erosion and submersion, such as dikes, cliffs, and dune systems, - Maritime infrastructure such as wind farms, bridge piers and other offshore platforms. - Terrestrial applications such as soil stabilization, bank stabilization, slope stabilization, embankment stabilization, and limestone consolidation, - Protective structures such as foundations and retention basins, - Protection against natural phenomena such as erosion and flooding, - The stabilization of infrastructure such as port facilities and concrete-based infrastructure.

[0128] Materials rehabilitation

[0129] For the purposes of this invention, "materials rehabilitation" means any application of the process aimed at repairing, enhancing or transforming materials or substrates in a controlled environment, separate from their place of origin or final use.

[0130] For example, the rehabilitation of materials includes: • Repair applications such as the restoration of damaged materials, • Production of materials such as building blocks, prefabricated structures and composite materials.

[0131] Materials design

[0132] For the purposes of the present invention, "materials design" means any application of the process aimed at forming new materials by consolidating substrates in a controlled environment, separate from their place of origin or final use.

[0133] Examples include the manufacture of gabions to replace the extraction and transport of rock blocks, the production of bricks from sewage sludge, decorative objects for aquariums, etc. Examples

[0134] Example 1 - Isolation, selection and characterization of biocalcifying bacteria

[0135] In order to limit the use of exogenous bacteria to the environment to be consolidated, it is necessary to isolate and select biocalcifying bacteria close to the study site.

[0136] Initially, aseptic samples of water, soil or sediment must be taken from near the study area.

[0137] In a second step, these different samples must be placed in a medium adapted to bacterial growth and enriched with calcium in order to select biocalcifying bacteria exhibiting mineral deposits on the surface of the bacterial colonies formed.

[0138] In a third step, the mineral deposits are characterized by Raman microspectrometry and X-ray diffraction in order to validate the presence of mineral phases based on calcium and / or magnesium whose precipitation has been induced by bacteria.

[0139] In a fourth step, the metabolism(s) of the biocalcifying bacteria thus selected are characterized by enzymatic tests: i) urea-indole medium to demonstrate urease activity with induction of a color change of the medium when urea is degraded by urease; ii) medium with para-nitrophenol acetate which under the action of carbonic anhydrase is transformed into para-nitrophenol (yellow compound), to demonstrate carbonic anhydrase activity.

[0140] Bacteria capable of inducing the precipitation of mineral phase based on calcium and / or magnesium in the absence of urea will be selected.

[0141] Identifying these bacteria by 16s rDNA sequencing also provides information on their metabolic potential and confirms the results obtained by enzymatic tests.

[0142] This selection process led to the identification of 14 biocalcifying bacteria from the marine environment of the Atlantic coast near La Rochelle. Raman microspectrometry and X-ray diffraction confirmed the precipitation of magnesium calcite-type CaCO₃ by these bacteria. All 14 bacterial strains possess the carbonic anhydrase pathway for biocalcification.

[0143] The isolated strains were designated CDi (i=1 to 10) for bacteria taken from the limestone deposit (CD), Md1 for bacteria from the mud of the Angoulins coast, and SWi (i=1 to 3) for bacteria from seawater sampled in the port of La Rochelle or on the Angoulins coast.

[0144] The 14 biocalcifying bacterial strains were identified by sequencing the 16S rDNA gene and comparison with the sequences of GenBank, SILVA ACT and RDP II. All sequences corresponded to at least one identified strain with more than 98.5% identity.

[0145] Based on their phylogenetic relationships, the isolates were divided into three groups: Gammaproteobacteria (50%), Alphaproteobacteria (21.4%) and Firmicutes (28.6%).

[0146] Within the Gammaproteobacteria class, six isolates (MD1, SW1 to SW3, CD9 and CD10) were assigned to the genus Pseudoalteromonas and CD3 to the species Pseudidiomarina maritima. The Gammaproteobacteria group, which comprised 7 of the 14 isolated bacterial strains, was the largest.

[0147] The Alphaproteobacteria comprised three isolates (CD1, CD4, and CD5), all of which were assigned to the species Epibacterium motile. The Firmicutes comprised four isolates (CD2, CD7, CD6, and CD8), where CD2 and CD7 were most closely related to Bhargavaea sp., and the two isolates CD6 and CD8 were affiliated to the species Virgibacillus halodenitrificans and Pianococcus maritimus, respectively.

[0148] Thus, mainly Gram-negative bacilli (71.4%, Gammaproteobacteria and Alphaproteobacteria) were identified, but also Gram-positive bacilli (21.4%, Firmicutes) and Gram-positive cocci (7.1%, Firmicutes), microorganisms which are phylogenetically quite distant but all capable of biomineralization.

[0149] Thus, the 14 bacterial strains are as follows: Among the gammaproteobacteria: MD1 Pseudoalteromonas sp. SW3 Pseudoalteromonas sp. SW1 Pseudoalteromonas sp. SW2 Pseudoalteromonas sp. CD10 Pseudoalteromonas sp. CD9 Pseudoalteromonas sp. CD3 Pseudomarine maritima Among the alphaproteobacteria CD5 Epibacterium motile CD1 Epibacterium motile CD4 Epibacterium mobile Among the firmicutes CD6 Virgibacillus halodenitrificansCD8 Pianococcus maritimus CD7 Bhargavaea beijingensis CD2 Bhargavaea ginseng.

[0150] This work was described in Vincent, J.; Sabot, R.; Lanneluc, I.; Refait, P.; Turcry, P.; Mahieux, P.-Y.; Jeannin, M.; Sablé, S. Biomineralization of Calcium Carbonate by Marine Bacterial Strains Isolated from Calcareous Deposits. Matériaux & Techniques 2020, 108, 302, doi:10.1051 / mattech / 2020027, and Vincent, J.; Colin, B.; Lanneluc, I.; Sabot, R.; Sopéna, V.; Turcry, P.; Mahieux, P.-Y.; Refait, P.; Jeannin, M.; Sablé, S. New Biocalcifying Marine Bacterial Strains Isolated from Calcareous Deposits and Immediate Surroundings. Microorganisms 2022, 10, 76, doi:10.3390 / microorganisms10010076.

[0151] Example 2 - Evaluation of the ability of endogenous biocalcifying bacteria to consolidate an initially non-cohesive granular substrate by creating a calcareous network

[0152] An experimental setup was set up and is shown in [Fig. 1]. Similar methods have also been described in the literature (Bernardi et al. 2014; Harkes et al. 2010; Henze and Randall 2018).

[0153] The column system consists of a PVC cylinder (commercially available PVC pipe used for plumbing) with an internal diameter of 4.45 cm and a length of 15 cm. Two PVC sleeves are glued to each end of the column, allowing for the attachment of lids. The lower and upper lids (also made of PVC) are identical and removable, both screwed onto the sleeves. Two O-rings ensure the system is watertight. The lids are drilled to accommodate a 0.5 cm diameter tubular connector to allow the cementing solution to circulate through the column. In addition, two membranes (an abrasive pad such as a Scotch-Brite pad and a paper filter such as a coffee filter), each 4.4 cm in diameter, are placed on either side of the column to prevent the granular substrate from escaping the mold through the percolation tubes, as described by Henze et al. (Henze and Randall 2018).All PVC parts of the assembly were previously cleaned and disinfected with 70° ethanol.

[0154] Washed, 100% silica (SiO2) alluvial sand with a grain size of 0 / 4 mm is used as the granular substrate. The use of 100% silica sand aims to clearly identify the calcareous cement within the agglomerate using analytical techniques (optical microscopy, Raman microspectrometry, and X-ray micro-tomography). The sand was previously sterilized at 115°C for 20 minutes. Other initially non-cohesive granular substrates, as well as different geometries and volumes, could be adapted to this experimental setup.

[0155] Regardless of the setup, an isolated or consortium culture of biocalcifying bacteria is carried out in 100 mL of culture medium suitable for the bacteria used, with the appropriate temperature and incubation time. A 10x concentrated and / or lyophilized bacterial culture, as well as the use of enzymes involved in biocalcification, can also be employed in this system.

[0156] Before being placed in the column, the sand underwent pretreatment, similar to that described by Henze et al. (Henze and Randall 2018), consisting of adding a volume of sand slightly greater than the total volume of the column (250 cm³). 3sand) in the bacterial culture, then the sand / bacteria mixture is slowly agitated (60 rpm) for a sufficient time to allow for the "reactivation / rehydration" of the bacteria (in the case of lyophilized bacteria) and their adhesion to the sand grains, resulting in their homogeneous distribution within the sandy granular skeleton. In our case, this corresponds to 4 hours at 30°C. The sand / bacteria mixture is then placed in the column. Excess liquid culture medium is removed through the system's bottom outlet. The column is then hermetically sealed at both ends and connected via a peristaltic pump to a bottle containing a sterile "cementing" solution. From the bottom of the column, 60 ml of cementing solution is injected every 3 hours at a rate of 10 ml / min. -1(conditions adaptable according to experimental conditions), at room temperature, for one month as described by Bernhardi et al (Bernardi et al. 2014).

[0157] In the present study, the cementation solution is composed of 0.5 gL of nutrient broth -1 , of CaCh 3.7 gL -1 (25 mmol.L -1 ) and urea 20 gL -1 (330 mmol.L -1The columns are prepared in reverse osmosis water or sterile natural seawater for marine bacteria. The treatment time may vary depending on the experiment. At the end of the experiment, the columns are dried in an oven at 60°C for 3 days. They are then cut lengthwise to extract the formed bio-bricks. These bio-bricks are placed in an oven at 100°C for 24°C before mechanical and physical analyses, which require a thoroughly dried material. Thermogravimetric, axial compression, and water porosity analyses are used to characterize the bioformed concretions and thus select the most efficient endogenous biocalcifying bacteria.

[0158] The experimental setup was tested and validated with the ureolytic strain Sporosarcina pasteurii DSM 33, a strain of terrestrial origin already used for applications in biocalcification (Henze and Randall 2018; Bernardi et al. 2014).

[0159] The setup also showed that 13 of the 14 isolated marine biocalcifying bacteria have the ability to form, in the presence of urea, sandy-calcareous agglomerates under these experimental conditions (marine condition) in 1 month of treatment.

[0160] The 13 bacteria are as follows: - MD1 Pseudoalteromonas sp. - SW3 Pseudoalteromonas sp. - SW1 Pseudoalteromonas sp. - SW2 Pseudoalteromonas sp. - CD 10 Pseudoalteromonas sp. - CD9 Pseudoalteromonas sp. - CD3 Pseudidiomarina maritima - CD5 Epibacterium mobile - CD1 Epibacterium motile - CD4 Epibacterium motile - CD6 Virgibacillus halodenitrificans - CD8 Pianococcus maritimus - CD2 Bhargavaea ginsengi.

[0161] Based on physicochemical analyses, three biocalcifying bacterial strains were identified as the most effective in consolidating sand, yielding a material with an axial compressive strength exceeding 3 MPa, a final porosity as low as 15%, and a high CaCO₃ content of up to 12%. These three bacterial strains were selected: - CD4 Epibacterium motile - MD1 Pseudoalteromonas sp. - CD8 Pianococcus maritimus Furthermore, the above setup was adapted and validated on a smaller scale using a 20 mL syringe-type PVC cylinder (2 cm in diameter and 8 cm long), thus demonstrating the consolidation of the sandy substrate using a cementation solution composed of natural seawater, sterilized by filtration, enriched with calcium and urea, and fed into the system in a continuous flow. The nutrients naturally present in the seawater were sufficient to promote bacterial biocalcification.

[0162] Example 3 - Process for consolidating a substrate using biocalcifying bacteria under cathodic polarization conditions

[0163] The biocalcification / biocementation capacity of bacteria is evaluated under cathodic polarization conditions. The biocementation device under cathodic polarization consists of a vertical assembly comprising one or more 20 mL syringes with electrodes connected to a potentiostat for cathodic polarization (Figure 2). The entire assembly is connected to a peristaltic pump that delivers a cementation solution from bottom to top with a continuous or intermittent flow rate.

[0164] All so-called marine and non-marine biocalcifying bacterial strains capable of inducing CaCO3 precipitation can be used in isolation or in consortium in this setup, as well as concentrated and / or lyophilized bacteria and / or enzymes involved in biocalcification.

[0165] A bacterial culture is carried out in 10 mL of culture medium suitable for the strain(s) used, at the optimal growth temperature and for the appropriate duration. For inoculation of the sand with the bacteria, 40 g of previously sterilized silica sand (115°C for 20 min) are mixed with this bacterial culture and agitated slowly (60 rpm) for 4 to 6 hours at the optimal growth temperature to promote bacterial adhesion to the sand and ensure homogeneous distribution.

[0166] For cathodic protection, a carbon steel spiral, acting as the cathode and connected to an electrical wire, is placed inside the syringe. The sand-bacteria mixture is then added to the syringe. The anode, a platinum filament, is positioned above the Scotch-Brite type filter in the upper part of the setup. To secure the assembly and minimize contamination, the syringe plunger is positioned at the top of the syringe.

[0167] From the bottom of the column, a cementation solution, composed of natural seawater sterilized by filtration and enriched with 25 mM CaCh, is continuously injected at a low flow rate (approximately 0.03 mL / min). -1 ), at room temperature, until the end of the experiment.

[0168] The cementation solution may contain an enrichment in nutrient broth.

[0169] Advantageously, the urea supply has been removed because cathodic polarization also allows the medium to be alkalized and can therefore replace the use of urea.

[0170] A cathodic polarization between the cathode and the anode is established by an imposed current density of -150 pA.cm -2 based on a continuous or sequential polarization cycle (every 6 hours).

[0171] At the end of the experiment, the syringes are placed in an oven at 60°C for 3 days. They are then cut open to extract the bio-bricks that have formed. These bio-bricks are then placed in an oven at 100°C for 2 or 3 days before mechanical and physical analyses, which require a dry material.

[0172] Thermogravimetric, axial compression and water porosity analyses allow the concretions formed to be characterized.

[0173] Polarizing steel in an electrolyte induces electrochemical reactions that locally increase the pH, allowing brucite and calcium carbonate to precipitate on the surface of the polarized steel. Concurrently, the action of biocalcifying bacteria provides a greater quantity of hydrogen carbonate (HCO3). -by hydration of CO2 under the action of carbonic anhydrase. The alkaline conditions provided by cathodic polarization then allow the conversion of hydrogen carbonates HCO3 - in CO3 2- which precipitate into CaCO3 in the presence of Ca 2+ , originating from both the cell and the environment. This action of cathodic polarization concomitant with that of the bacteria makes it possible to overcome the alkalinization caused by the hydrolysis of urea by ureloytic bacteria.

[0174] The consolidation process using biocalcifying bacteria under cathodic polarization conditions was implemented with a 10-fold concentrated, lyophilized marine bacterial strain (CD4 motile Epibacterium) for one month of treatment with continuous circulation in a cementation solution (flow rate = 0.03 mL / min). -1 ) composed of natural seawater sterilized by filtration, enriched only with CaCh 25 mmol.L -1, with an imposed current density of -150 pA.cm -2 based on a sequential polarization cycle (every 6 hours).

[0175] Advantageously, the process according to the invention made it possible to obtain a thicker calcomagnesian agglomerate (Figure 3B) compared to a calcomagnesian agglomerate that would be obtained under cathodic polarization conditions only (Figure 3A).

[0176] Example 4: Biocementation device

[0177] As an alternative to the device described in Example 3 and illustrated in Figure 2, a 3D-printed assembly using PLA (polylactic acid) filaments can be used for greater design and adaptation flexibility. This device is illustrated in Figure 4.

[0178] A cylindrical basket, made of PLA, was developed to serve as internal support for all the materials used in the experiment (cathodic, anodic, and salt bridge compartments), with a hollow cannula at one end to ensure the circulation of the cementation solution through the column. This component is positioned inside a prefabricated 500 mL pharmaceutical syringe.

[0179] 2) A "puck" was also designed using 3D printing. Its structure was perforated on both sides, with the addition of channels that were cut into both faces to ensure a more homogeneous distribution of the cementation solution within the device. This device also prevents the granular substrate from escaping the structure through the percolation tubes, which are themselves connected to the cannula of the PLA "cylindrical basket."

[0180] The column system now consists of a 3D-printed PLA cylinder with an inner diameter of 6.4 cm and a length of 19 cm, and a 6 cm diameter puck placed at the bottom of the column (Figure 1). Once these two elements are in place, a coil made of carbon steel wire (acting as the cathode), with a diameter of 4 cm and a height of 5.5 cm, is positioned inside the column (cathodic compartment). This coil is then immersed in 100% silica (SiO2) alluvial sand with a grain size of 0 / 4 mm. Biocalcifying bacteria are also introduced into the cathodic compartment, within the granular medium, to trigger the biocalcification processes coupled with polarization. The experimental setup could also be adapted to other initially non-cohesive granular substrates, as well as to variable geometries and volumes.

[0181] The cementing solution is then circulated from bottom to top using a peristaltic pump until it reaches the top of the sand column. A salt bridge layer (15 gL) is then applied. -1 of agar-agar and 25 gL -1 of KNO3) is then deposited on the surface of the damp sand. Finally, a 25 gL salt water solution is applied. -1 NaCl is deposited above the salt bridge. A platinum wire (anode) is immersed in it to form the anodic compartment.

[0182] A cathodic polarization is then applied between the cathode and the anode.

[0183] Example 5: Evaluation of different bacterial incorporation methods, cementation solution formulations, and cathodic polarization conditions

[0184] The bacterial strains Epibacterium motile CD4 and Pseudoalteromonas sp. MD1 were prepared differently depending on the test: Standard concentration liquid suspension (Liquid, 1X); Concentrated liquid suspension (Liquid, 10X); Freeze-dried incorporation into the sand (Freeze-dried with sand, 1X); or Concentrated and freeze-dried incorporation into sand (Freeze-dried with sand, 10X).

[0185] For the liquid bacterial supply (1X), a bacterial culture was prepared in 100 mL of Marine Broth (MB) culture medium for 24 hours at 30°C. The resulting suspension was then contacted with sterile sand for 4 hours under agitation at 30°C to promote cell adhesion and homogenization within the substrate.

[0186] For the 10X concentrated condition, 1 L of a 24-hour culture in MB medium was centrifuged to concentrate the cells to a final volume of 100 mL. This concentrated suspension was then incorporated into sterile sand according to the same protocol (4 hours with stirring at 30°C).

[0187] An alternative and innovative approach has also been developed, based on the direct freeze-drying of bacteria in a sandy substrate. In this case, 100 mL of bacterial culture (1X or 10X) was incorporated into 500 g of sterile sand. The mixture was then frozen at -80°C and subsequently freeze-dried. This method yields a ready-to-use material that is easier to store and handle, and directly incorporates the elements necessary for the process.

[0188] The intensity and application method of the current were also optimization parameters. Two configurations were tested: a cyclic current of -300 pA.cm -2 and a direct current of -150pA.cm -2 .

[0189] The different tests are listed in the table below:

[0190] [Table 2] Bacteria Code Nature of the input Cementation solution* Impressed current used in bacteria the test E1 CD4 Liquid, 1X Filtered EDMN + 0.5 gL 1 of BN + -300pA.cm -2 25mM cyclic CaCh 6h on / off E2 CD4 Liquid, 10X filtered EDMN + 0.5 gL 1 of BN + -300pA.cm -2 25 mM cyclic CaCh 6h on / off E3 MD1 Liquid, 1X filtered EDMN + 25 mM CaCh -150 pA.cm -2 Continuous E4 CD4 Lyophilized with filtered EDMN + 25mM CaCh -150pA.cm -2 continuous sand, 1X Lyophilized E5 CD4 with filtered EDMN + 25 mM CaCh -150 pA.cm -2 continuous sand, 10X E6 MD1 Lyophilized with filtered EDMN + 25mM CaCh -150pA.cm -2 continuous sand, 1X E7 MD1 Lyophilized with filtered EDMN + 25mM CaCh -150pA.cm -2 continuous sand, 10X

[0191] EDMN = Natural seawater. BN: Nutrient broth (composed of 10.0 g / L of Tryptone) -1 meat extract: 5.0 g. -1 L and Sodium Chloride: 5.0 gL -1), CaCh: calcium chloride

[0192] Different formulations of cementation solutions were evaluated. All consisted of filtered natural seawater (NSW) supplemented with CaCh (25 mM). Some formulations also included a nutrient supply in the form of nutrient broth (NB) at 0.5 g / L -1 , while others lacked it, in order to assess the influence of this addition on the process.

[0193] While all trials resulted in the formation of an agglomeration around the spring steel, trial E5 proved the most conclusive and is shown in Figure 5. It would appear that the preparation incorporating concentrated (10X) bacteria directly freeze-dried into the granular substrate represents a particularly promising approach for the future. The other trials also confirmed that the addition of nutrients was not necessary for the process to proceed correctly and that a direct current of -150 pA.cm was sufficient. -2 was sufficient to promote the formation of agglomerates.

[0194] Example 6:

[0195] In parallel with the biocementation trials, growth tests were carried out on the two most promising bacterial strains, CD4 (Epibacterium motile) and MD1 (Pseudoalteromonas sp.), over a period of 7 days. These experiments were conducted in different culture media, with monitoring of the culture pH, monitoring of bacterial growth by cell count, and measurement of the CaCO₃ level formed by thermogravimetric analysis (TGA) (Figure 6).

[0196] These trials pursued several objectives, namely: Evaluate CaCO₃ production in non-marine environments for adaptation to soils or other non-marine substrates. Compare the effect of different calcium sources, including calcium chloride (CaCh) and calcium acetate (Ca(CH3COO)2), and To show that the bicarbonate supply required for the formation of CaCO₃ is due to the metabolic activity of biocalcifying bacteria, particularly under conditions without exogenous bicarbonate supply.

[0197] The culture media tested included: C-CaCh Physiological Water: Physiological water supplemented with CaCh, composed of 9g of NaCl. 1 , NaHCOs 0.16g.L -1 , yeast extract 1 gL -1 , proteose / peptone (sigma) 5 gL -1 and of CaCh.2H2O 5.47 gL 1 (37.2 mM). MB-CaCh: MB supplemented with CaCh, composed of Marine broth (MB) culture medium (Condalab) and CaCh.2H2O 3.67 gL 1 (25 mM). Phy-CaCh Water: Physiological water without bicarbonate and supplemented with CaCh, composed of 9g NaCl. 1 , yeast extract 1 gL -1 , proteose / peptone (sigma) 5 gL -1 and CaCl2.2H2O 5.47 gL 1 (37.2 mM). Physiological water (CaAc): Physiological water supplemented with calcium acetate, composed of NaCl 9g / L, NaHCO3 0.16g / L -1 , yeast extract 1 gL -1 , proteose / peptone (sigma) 5 gL -1 and calcium acetate 6.55 gL -1 (37.2 mM). MB-Ac.Ca: MB supplemented with calcium acetate, composed of Marine broth culture medium (Condalab) and 4.4 gL of calcium acetate -1 (25 mM). Comparison between marine (MB) and non-marine (physiological water) environments shows that, for the CD4 bacterial strain, bacterial growth and pH evolution are relatively similar in both environments. In contrast, the MD1 strain exhibits more limited growth in non-marine environments, while still retaining the ability to induce the precipitation of a certain amount of CaCO₃. For both biocalcifying strains, the amount of CaCO₃ bioprecipitated appears to be generally greater in marine environments. These results also demonstrate, for both strains tested, that the use of calcium acetate, as a substitute for CaCl2, promotes higher CaCO3 precipitation, particularly in non-marine environments where more alkaline pH values ​​were observed. It is likely that the acetate is metabolized by the MD1 strain, which could explain its improved growth under these conditions. Finally, a comparison of non-marine environments (physiological water) with and without bicarbonate supplementation shows similar amounts of bioprecipitated CaCO₃. These observations suggest that the bicarbonates required for CaCO₃ precipitation originate primarily from the metabolic activity of biocalcifying bacteria, confirming their direct role in the biomineralization process. These results demonstrate that the tested strains retain the capacity for calcium carbonate bioprecipitation in non-marine environments, confirming their potential for applications in soils or other non-marine substrates. They also show that the nature of the calcium source influences precipitation yield, with calcium acetate proving to be a particularly favorable source in certain applications. Finally, they demonstrate that an exogenous supply of bicarbonate is not necessarily required for CaCO₃ formation, as bicarbonate species can be generated, at least in part, by the metabolic activity of the biocalcifying bacteria.

Claims

Demands

1. In situ substrate consolidation process comprising the steps of: i) Seeding a medium comprising the substrate with biocalcifying bacteria ii) Application of cathodic polarization with an imposed current density.

2. Ex situ substrate consolidation process comprising the steps of: i) Mixing biocalcifying bacteria with a substrate, ii) Adding a cementation solution to the mixture obtained in step i) iii) Applying cathodic polarization with an imposed current density.

3. Consolidation process according to claim 1 or 2, wherein biocalcifying bacteria induce the precipitation of calcium carbonate (CaCOs) via carbonic anhydrase.

4. Consolidation method according to any one of claims 1 to 3, wherein cathodic polarization is applied on a continuous or sequential polarization cycle, preferably on a sequential cycle, and preferably on a continuous polarization cycle.

5. A consolidation method according to any one of claims 1 to 4, wherein the cathodic polarization is a cathodic polarization with an imposed current density, preferably applied with a current density between -10 pA.cirr 2 and -600 pA.cnrr 2 , preferably between -125 pA.cirr 2 and -175 pA.cirr 2 , and preferably, in which cathodic polarization is applied with a current density of -150 pA. 2 .

6. Consolidation process according to any one of claims 1 to 5, wherein the substrate is selected from a powdery substrate, a cohesive substrate or a mixture of powdery and cohesive substrates, and is preferably selected from sand, mud, sediments, glass beads, clay, gravel, limestone, industrial sludge, mining tailings, ash, silt, peat and inert industrial waste.

7. Consolidation process according to claim 1 comprising a preliminary step of selecting biocalcifying bacteria from the medium and preferably, endogenous bacteria from the medium.

8. Consolidation method according to claim 1, wherein the medium is the cementation solution.

9. Consolidation method according to any one of claims 1 to 7, wherein a cementation solution is added to the medium.

10. A method according to claim 9, wherein the cementing solution comprises seawater or physiological water, preferably seawater, and may further comprise at least one nutrient selected from calcium, magnesium, organic compounds such as glucose, CO2, nitrogenous compounds such as urea, ammonium salts, nitrates, nitrites, amino acids, peptides and proteins, phosphate, potassium and mixtures thereof.

11. Consolidation process according to any one of the preceding claims, wherein the cementation solution does not contain exogenous urea.

12. A method according to any one of the preceding claims, wherein the cementing solution is injected in a continuous or discontinuous flow through the substrate, and preferably at a flow rate of between 0.001 and 1000 mL / min -1 , preferably 0.01 and 0.1 mL.min -1 , preferably between 0.02 and 0.05 mL.min -1 and preferably at approximately 0.03 mL / min -1 .

13. Consolidation process according to any one of claims 1 to 12, wherein the biocalcifying bacteria are selected from gammaproteobacteria, alphaproteobacteria, firmicutes and mixtures thereof and preferably wherein the gammaproteobacteria are selected from Pseudoalteromonas sp. and Pseudidiomarina maritima, Alphaproteobacteria are chosen from Epibacterium mobile and firmicutes are chosen from Virgibacillus halodenitrificans, Pianococcus maritimus, Bhargavaea beijingensis, and Bhargavaea ginsengi, and even more preferentially biocalcifying bacteria are chosen from MD1 Pseudoalteromonas sp., SW1 Pseudoalteromonas sp., SW2 Pseudoalteromonas sp., SW3 Pseudoalteromonas sp., CD9 Pseudoalteromonas sp., CD 10 Pseudoalteromonas sp., CD3 Pseudidiomarina maritima, CD1 Epibacterium mobile, CD4 Epibacterium mobile, CD5 Epibacterium mobile, CD6 Virgibacillus halodenitrificans, CD8 Pianococcus maritimus, CD2 Bhargavaea ginsengi, CD7 Bhargavaea beijingensis and their mixtures and preferably, the biocalcifying bacteria are chosen from CD4 Epibacterium mobile, MD1 Pseudoalteromonas sp., CD8 Pianococcus maritimus and their mixtures^.

14. Consolidation process according to any one of claims 1 to 13, wherein the biocalcifying bacteria are used as such or in concentrated and / or lyophilized form.

15. Use of a consolidation process according to any one of claims 1, 3 to 14, for environmental geotechnical stabilization, and preferably for coastal and port protection to combat coastal erosion and submersion such as dikes, cliffs, dune ridges, marine infrastructure such as wind farms, bridge piers and other offshore platforms, land applications such as soil stabilization, banks, slopes, embankments and consolidation of limestone, protective works such as foundations and retention basins, protection against natural phenomena such as erosion and flooding, stabilization of infrastructure such as port works and concrete-based infrastructure.

16. Use of a consolidation process according to any one of claims 2 to 6 and 11 to 14, for the rehabilitation or design of materials, and preferably for the repair of materials such as the restoration of damaged materials, the production of materials such as building blocks, prefabricated structures and composite materials.

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