Process of growing a biofilm

Abstract

The present invention relates to a process of growing a biofilm on a glass matrix comprising a step of contacting glass fragments with at least one microorganism in an open flow reactor.

Description

[0001] PROCESS OF GROWING A BIOFILM

[0002] Field of the invention

[0003] The present invention concerns a process of growing a biofilm directly on a glass matrix, the use of this process for extracting metals from glasses and in the context of glass recycling processes. The present invention also relates to glasses on which biofilms have grown and which have been subsequently recovered.

[0004] Background of the invention

[0005] Glass is a material widely used in various industrial, domestic and medical sectors. As a result, glass waste represents a significant proportion of the solid waste produced annually. Traditional glass recycling, although widely practiced, has significant limitations, including high energy consumption and degradation of recycled glass quality over the cycles. In addition, conventional recycling processes do not allow the recovery of certain chemical compounds present in the glass matrix, such as metals, which may be of significant value or potentially harmful.

[0006] Consequently, there is a need for new approaches to the targeted extraction of certain chemical compounds from glass, so that the glass can be reused in a variety of applications, and the extracted chemical compounds can be safely recovered or reused.

[0007] It is known that certain micro-organisms, notably biofilm-forming bacteria, are capable of interacting with mineral surfaces, including glass.

[0008] However, existing methods for growing biofilms in the presence of glass substrates are mainly carried out in closed environments, such as liquid cultures. Although these methods enable bacterial growth and biofilm formation, they have several drawbacks, notably a poor attachment of the biofilm to the glass surface linked to the progressive enrichment of the liquid solution with nutrients from the glass, subjected to dissolution, and consequently less direct interactions between the glass and the bacteria. Moreover, working in closed systems does not allow long-term experiment, as the nutrients provided by the nutritive solution are progressively consumed by bacteria, which rapidly reach a stationary phase, followed by a death phase. As a result, there is no process that takes advantage of the interaction between biofilms and mineral surfaces, particularly glass, to optimize the extraction of chemical compounds from the surface, notably in the context of glass recycling.

[0009] Thus, there is a need for an ecologically and economically viable alternative to conventional glass recycling methods, while at the same time providing a new route for the targeted extraction of chemical compounds.

[0010] Summary of the invention

[0011] The present invention arises from the unexpected finding by the inventors that the implementation of a process of growing a biofilm on glass samples in aqueous media with open flow and constant renewal of the culture medium, makes it possible to obtain a thick biofilm on the surface of the glass samples. The inventors have demonstrated that this biofilm has good adhesion and uniformity on the glass samples. In addition, the process developed by the inventors also makes it possible to work over long periods of time by the continuous renewal of substrates and nutrients required for microbial activity. The inventors have also demonstrated that this process is particularly suitable for extracting chemical compounds from glass.

[0012] Thus, the present invention relates to a process of growing an adherent biofilm on a glass matrix comprising a step of contacting glass fragments with at least one microorganism in a reactor, in particular an open flow reactor.

[0013] The present invention also relates to a process for extracting at least one metal from glass comprising carrying out the process as defined above.

[0014] The present invention also relates to a glass obtained by the process as defined above.

[0015] The present invention also relates to a process of recycling a glass comprising the following steps:

[0016] - Carrying out the process as defined above;

[0017] - Recovery of the glass;

[0018] - Recycling the glass.

[0019] Detailed description of the invention

[0020] Definition

[0021] In the specification and in the claims, the terms “including”, “comprising” and “containing” can be used interchangeably. These terms are open-ended terms and should be interpreted to mean “including”, but not limited to. Thus, when an object “comprises” or “contains” one or several elements, other elements than those mentioned may also be included in the object. These terms encompass the more restrictive terms “consisting essentially of” and “consisting of.” When an object is said to “consist of” one or several elements, the object is limited to the listed elements and cannot include other elements than those mentioned.

[0022] Bio film

[0023] Microorganisms, in particular bacteria, often organize themselves in biofilms. The term “biofilm” as used herein refers to a community of microorganisms adhered to a surface and covered or encapsulated by a matrix of extracellular polymeric substance (EPS) that is produced by the members of this community. Typically, the matrix is composed of polysaccharides, proteins, lipids, and nucleic acids.

[0024] Biofilms according to the present description can be composed of a singlespecies microorganism or can be composed of several microorganisms species.

[0025] Typically, the formation of a biofilm on a surface takes place in four main stages: (i) the first stage is initiated by the development of a cluster of microorganisms, such as bacteria, in planktonic form (i.e. within a film of water on the surface of the material); (ii) this cluster progressively adheres to the surface; (iii) the microorganisms, such as bacteria, progressively organize themselves into microcolonies; (iv) a final stage corresponds to the maturation phase where a matrix of extracellular polymeric substance (EPS) is formed.

[0026] Preferably, the microorganisms is selected from the group consisting of bacteria, fungi, algae, protozoa and mixture thereof. More preferably, the microorganism according to the present description is at least one bacteria.

[0027] The bacteria can be chosen among any suitable bacteria well known to the person skilled in the art. In a preferred embodiment, the bacteria is selected from the group consisting of bacteria of the genus Bacillus, Arthrobacter, Paenibacillus, Gloeocapsa, Nostoc, Streptomyces, Pseudomonas, Burkholderia, Agrobacferium, Sphingomonas, Acinetobacter, and mixtures thereof. Preferably, the bacteria is selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, Bacillus thuringiensis, Arthrobacter crystallopoietes, Paenibacillus polymyxa, Paenibacillus vortex, Paenibacillus larvae, Paenibacillus alvei, Gloeocapsa magma, Gloeocapsa atrata, Nostoc commune, Nostoc punctiforme, Streptomyces griseus, Streptomyces albus, Pseudomonas aeruginosa. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas luteola, Burkolderia cepacian, and mixtures thereof.

[0028] Preferably, the biofilm according to the present description is formed directly on the glass matrix, i.e., it is formed in immediate contact with the glass surface, without any intermediate layers or barriers between the microorganisms and the glass. In other word, the biofilm attach and grow straight onto the glass substrate, using it as the foundational surface for biofilm development.

[0029] Preferably, biofilm formation on glass matrix according to the present description is fast. The first phase of development of a microorganism cluster, in particular a bacteria cluster, in plaktonic form occurs a few minutes, preferably few hours, such as from 30 minutes to 24 hours, from 1 hour to 24 hours, from 5 hours to 24 hours, from 10 hours to 24 hours, from 15 hours to 24 hours, after the at least one microorganism has been brought into contact with the glass fragments.

[0030] Preferably, after a few hours, preferably between 10 and 24 hours, more preferably in approximatively 24 hours, following contact between the at least one microorganism and the glass fragments, biofilm can be observed on the glass matrix.

[0031] Preferably, a biofilm comprising a community of microorganisms and a network of extracellular polymeric substances adhering to the glass matrix is formed in approximatively 24 hours from the time the at least one microorganism is brought into contact with the glass fragments according to the present description. Preferably, a biofilm is formed on the glass matrix between 15 hours and 30 hours, preferably between 18 hours and 30 hours, between 20 hours and 30 hours, or between 15 hours and 24 hours, between 18 hours and 24 hours, between 20 hours and 24 hours from the time the at least one microorganism is brought into contact with the glass fragments according to the present description.

[0032] Preferably, the biofilm has a thickness of between 2 and 200 pm, for example between 2 and 180 pm, between 2 and 160 pm, between 2 and 160 pm, between 2 and 140 m, between 2 and 120 pm, between 2 and 100 pm, between 2 and 80 pm, preferably after at least 1 day of culture. Biofilm thickness can be measured by any technique well known to the skilled person. For example, biofilm thickness can be measured using a scanning electron microscope (SEM).

[0033] The cultivation time can be adapted by the person skilled in the art. The cultivation time is preferably at least 1 day, at least 2 days, at least 3 days, at least 4 days at least 5 days. The cultivation time can last several months, for example the cultivation time can last 12 months, 1 1 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month.

[0034] Advantageously, the chemical compounds contained in the glass, such as for example phosphorus, iron, and manganese, can be used by the microorganisms, in particular the bacteria, to trigger microorganisms growth and the production of biofilms clinging to the glass surface.

[0035] Preferably, the biofilm according to the present description is uniform, i.e. it has a consistent thickness throughout, without significant variations, gaps, or irregularities. Preferably also, the biofilm uniformly covers the entire surface area of the glass matrix.

[0036] Preferably, the biofilm has a firm attachment to the glass surface. Without wishing to be bound by any theory, this strong adhesion may be mediated by extracellular polymeric substances (EPS) which bind the biofilm to glass fragments. In addition, surface of the glass fragments undergo changes that may a Iter their surfaces due to the formation of the biofilm. Such changes can create a rougher, more porous surface as well as micro-niches that can provides a better grip for the biofilm and enhance its ability to cling tightly.

[0037] Glass matrix

[0038] A glass matrix in the context of the present description refers to a structural network made of glass fragments that provides a surface for microorganisms to attach to, grow, and form biofilms.

[0039] Glass fragments according to the present description can come from any glass known to the skilled person. Preferably, the glass fragments are selected from the group consisting of fragments of soda glasses, soda-lime glasses, borosilicate glasses, lead glasses, silica glasses, silico-calco-potassium glasses, glass-ceramics, aluminosilicate glasses, quartz glasses, mixed alkali glasses, potassium glasses, calcium-potassium glasses, and mixtures thereof.

[0040] Preferably, glass fragments according to the present description comprise at least one element selected from the group consisting of aluminum, calcium, magnesium, manganese, potassium, lead, chromium, cobalt, copper, sodium, iron, selenium, silicon, boron, phosphorus, germanium and arsenic in particular in oxide, selenides, sulfides, and fluorides form. Preferably, glass fragments according to the invention comprise at least one oxide selected from the group consisting of SiCh4-, MnO, FeO, SIO2, AI2O3, MgO, CaO, Na2O, K2O, B2O3, TIO2, ZnO, CuO, CU2O, NiO, U2O, CoO, La2C>3, ZrC>2, PbO, P2O5, and mixtures thereof.

[0041] Glass fragments can come from a variety of sources. They may, for example, come from everyday objects, industrial production waste, construction and demolition waste, laboratory and medical glasses, optical lenses, optical fibers, bioglass or vitrification of nuclear waste etc.

[0042] Examples of objects used to supply glass fragments include bottles, jars, culinary glassware, glassware, dishes, production scrap from glass manufacturing plants, such as excess material, defective items or glass scraps, glass fragments from industrial processes using glass, such as glass fiber production, old windows, glass from demolished or renovated buildings, stained glass, glass materials from scientific and medical laboratories, light bulbs, glass fragments from art glassware, glass used for television sets and electronics, halogen lamp tubes, optical elements and telescope mirrors.

[0043] In one embodiment, the glass fragments are irregularly shaped and may consist of a mixture of fragments of different sizes and shapes, such as oval, cubic, round, flat, etc. In particular, glass fragments of different lengths, widths and thicknesses can be used. According to another embodiment, the glass fragments are regularly shaped and of similar sizes. In another embodiment, glass fragments are composed of whole objects, such as bottles, glass art objects, glass utensils, etc.

[0044] In the case of glass fragments with a circular or spherical shape, the glass fragments according to the present description preferably have a diameter of between 1 mm and 100 cm, such as between 1 mm and 80 cm, between 1 mm and 60 cm, between 1 mm and 40 cm, between 1 mm and 20 cm, between 1 and 10 cm, between 1 mm and 8 cm, between 1 mm and 6 cm, between 1 mm and 4 cm, between 1 mm and 2 cm, between 1 mm and 1 cm.

[0045] According to one embodiment of the invention, the glass fragments have a length ranging from a few millimeters to several centimeters. Preferably, the glass fragments have a length of between 0.1 mm and 5 cm, between 0.5 mm and 5 cm, between 0.5 mm and 4 cm, between 0.5 mm and 3 cm, between 0.5 mm and 2 cm, between 0.5 mm and 1 cm, or between 1 cm and 100 cm, between 1 cm and 95 cm, between 1 cm and 90 cm, between 1 cm and 85 cm, between 1 cm and 80 cm, between 1 cm and 75 cm, between 1 cm and 70 cm, between 1 cm and 65 cm, between 1 cm and 60 cm, between 1 cm and 55 cm, between 1 cm and 50 cm, between 1 cm and 45 cm, between 1 cm and 40 cm, between 1 cm and 35 cm, between 1 cm and 30 cm, between 1 cm and 25 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, between 1 cm and 10 cm, between 1 cm and 5 cm.

[0046] According to one embodiment of the invention, the gloss fragments have a width ranging from a few millimeters to several centimeters. Preferably, the glass fragments have a width of between 0.1 mm and 5 cm, between 0.5 mm and 5 cm, between 0.5 mm and 4 cm, between 0.5 mm and 3 cm, between 0.5 mm and 2 cm, between 0.5 mm and 1 cm, or between 1 cm and 100 cm, between 1 cm and 95 cm, between 1 cm and 90 m, between 1 cm and 85 cm, between 1 cm and 80 cm, between 1 cm and 75 cm, between 1 cm and 70 cm, between 1 cm and 65 cm, between 1 cm and 60 cm, between 1 cm and 55 cm, between 1 cm and 50 cm, between 1 cm and 45 cm, between 1 cm and 40 cm, between 1 cm and 35 cm, between 1 cm and 30 cm, between 1 cm and 25 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, between 1 cm and 10 cm, between 1 cm and 5 cm.

[0047] Preferably, the glass fragments have a thickness of between 0.01 mm and 10 cm, for example between 0.1 mm and 8 cm, between 0.1 mm and 7 cm, between 0.1 mm and 6 cm, between 0.1 mm and 5 cm, between 0.1 mm and 4 cm, between 0.1 mm and 3 cm, between 0.1 mm and 2 cm, between 0.5 mm and 1 cm. Preferably, the glass fragments have a thickness of around 4 mm, 6 mm, 8 mm, 10 mm or 12 mm, 14 mm, 16 mm, 18 mm, 20 mm, 22 mm, 24 mm, 26 mm, 28 mm, 30 mm, 32 mm, 34 mm, 36 mm, 38 mm, or 40 mm.

[0048] Preferably, the surface of the glass fragments is comprised between 1.0 x 10“3m2and 5.0 x 10“3m2, such as between 1.5 x 10“3m2and 3.5 x 10“3m2, between 2.0 x 10“3m2and 3.0 x 10“3m2, or between 2.25 x 10“3m2and 2.75 x 10“3m2.

[0049] In one embodiment of the present description, glass fragments are in the form of glass powder. Glass powder can be a powder derived from the waste products of the glass manufacturing industry. Glass powder can also be obtained by reducing pieces of glass to very fine particles. Any method well known to the skilled person can be used to prepare glass powder. Examples of methods include mechanical grinding, wet grinding, dry grinding, cryogenic grinding etc.

[0050] Preferably, the granulometry of the glass powder according to the present embodiment is comprised between 10 and 1000 pm, such as for example between 10 and 900 m, 20 and 800 pm, 50 and 700 pm, 50 and 600 pm, 50 and 500 pm, 50 and 400 pm, 50 and 300 pm, or 50 and 200 pm. More preferably, the granulometry of the glass powder is comprised between 100 et 200 pm.

[0051] When biofilm forms on glass fragments, the glass layer in contact with the biofilm may undergo modifications and be altered.

[0052] These changes may involve the formation of cracks and micro-niches, as well as a rougher and / or more porous surface of glass. These features can be analyzed for example using techniques such as scanning electron microscopy (SEM) or atomic force microscopy (AFM).

[0053] In addition, these changes may also involve the release of elements from the glass, particularly alkaline, alkaline-earth and metal elements such as K, Ca, Mg, Mn, Al, Fe, Mn etc. The elemental composition of the glass may be altered, particularly if elements such as manganese or iron have been removed in significant quantities. Analysis by X-ray fluorescence spectroscopy (XRF) or SEM-coupled energy dispersive spectroscopy (EDS) can be used to determine residual chemical composition and quantify changes.

[0054] The process can also alter the oxidation state of elements present on the glass surface, which can be analyzed by techniques such as X-ray photoelectron spectroscopy (XPS).

[0055] The layer of weathered glass can be between 1 pm and 5 cm thick such as for example from 1 mm to 4 cm, from 1 mm to 3 cm, from 1 mm to 2 cm, or from 1 pm to 10 mm, from 1 pm to 9 mm, from 1 pm to 8 mm, from 1 pm to 7 mm, from 1 pm to 6 mm, from 1 pm to 5 mm, from 1 pm to 4 mm, from 1 pm to 3 mm, from 1 pm to 2 mm, from 1 pimto 1 mm.

[0056] Reactor

[0057] Preferably, microorganism according to the present description are cultured in a continuous flow of fresh medium while simultaneously removing waste products. Advantageously, fresh medium is continuously supplied to the open flow reactor, providing essential nutrients and maintaining optimal growth conditions. Spent medium is continuously removed, thus maintaining a stable environment.

[0058] As used herein, the term “open flow reactor” refers to a reactor where reactants such as fresh medium continuously flow into the reactor and products such as waste products flow out. In the present description, the terms “reactor” and “bioreactor” can be used interchangeably and refer to devices used for the cultivation of microorganisms under controlled conditions, with the supply of nutrients and the removal of waste products.

[0059] Typically, a bioreactor, in particular an open flow bioreactor, according to the present description comprises:

[0060] - A glass or stainless steel vessel or enclosure;

[0061] - A cap if required;

[0062] - at least one inlet for injecting a solution;

[0063] - optionally a stirring system;

[0064] - optionally sensors to measure temperature (thermometer), pH (pH meter), oxygen concentration, etc.

[0065] Reactor size, in particular open flow reactor size, according to the present description can vary from laboratory models ranging from 0.1 to 15 liters, to models used for industrialization testing (called “pilots”) ranging from 20 to 1 ,000 liters, to industrial production models that can exceed 1000 m3.

[0066] The person skilled in the art will know how to adapt the quantity of glass fragments to the type of reactor, in particular open flow reactor, used.

[0067] The solution renewal rate determines the hydraulic residence time, i.e. the time during which the solution, including suspended bacteria, remains in the reactor, in particular in the open flow reactor. In order to promote biofilm formation on glass fragments, it is preferable to minimize the proportion of bacteria in suspension. So, preferably, the residence time should be chosen to limit the growth of suspended bacteria. As an example, for P. putida bacteria, the time chosen is the time it takes to reach an exponential growth phase in the minimum nutrient medium, i.e. two days. Preferably, the renewal rate of the culture medium is adjusted to provide a residence time of the at least one microorganism suspended in the reactor, in particular in the open flow reactor, of at least two days.

[0068] Preferably, the flow rate of the culture medium injected into the reactor, in particular in the open flow reactor, can be adapted by the person skilled in the art, depending on the bacteria, the medium used and the size of the reactor. Preferably, the flow rate of culture medium injected into the reactor, in particular in the open flow reactor, is between 0.5 L / d and 50 L / d, for example from 0.5 L / d to 20 L / d, from 0.5 L / d to 10 L / d. In the case of large-volume reactors, e.g. from a few hundred to several thousand liters, the flow rate of the injected culture medium can be between 50 and 5000 L / d.

[0069] Preferably, the concentration of micro-organisms, in particular bacteria, is adjusted so as to have an optical density (OD) of between 1 and 2, preferably between 1 and 1.8, between 1 and 1.7, between 1 and 1.6, between 1 and 1.5, between 1 and 1.4, between 1 and 1.3, more preferably between 1 and 1.2. The optical density of the solution of micro-organisms, in particular bacteria, can be measured by any method well known to the person skilled in the art. For example, optical density can be measured using a spectrophotometer.

[0070] Preferably, the temperature inside the reactor, in particular in the open flow reactor, should be sufficient to ensure good bacterial growth. The temperature can easily be chosen by the person skilled in the art. Preferably, the temperature is between 10°c and 80°C, for example between 15 and 45°C, between 20 and 40°C, or around 30°C.

[0071] Preferably, the process is carried out in the presence of an excess of O2 in the reactor, in particular in the open flow reactor.

[0072] Preferably, the process is performed under stirring. Advantageously, stirring helps maintain good homogeneity of the solution.

[0073] Advantageously, the reactor conditions, in particular the flow rate, the temperature, the presence of O2, regulate microbial growth conditions, ensuring homogeneous, stable biofilm formation on the glass surface.

[0074] Advantageously, the conditions described above allow precise control of culture and contact conditions, enabling regulation of microbial growth and the efficiency of the extraction process.

[0075] Culture medium

[0076] Preferably, the culture medium according to the present description is sufficiently nutritious to support bacterial growth, but also sufficiently low in nutrients to force bacteria to cling to the glass surface.

[0077] In one embodiment, the culture medium is specifically designed to provide only essential nutrients that are not present in the glass, ensuring that the bacteria must interact with the glass to obtain all necessary elements for growth.

[0078] Preferably, the culture medium comprises at least one source of carbon and at least one source of nitrogen. In the case of glass fragments containing phosphorus, the culture medium preferably contains no added phosphorus, as the phosphorus required for bacterial growth is supplied directly from the glass fragments. Conversely, if the glass fragments do not contain phosphorus, the culture medium is preferably a minimal medium containing a low concentration of phosphorus to prevent excessive nutrient availability and encourage biofilm formation on the glass surface.

[0079] In one preferred embodiment, the culture medium is a minimum nutrient medium designed to act solely as a source of carbon and nitrogen for the bacteria. Since these two elements are generally not present in the composition of the glass, the medium does not supply them from other sources.

[0080] For instance, a specific minimum medium is composed of 1 g / L glucose as the carbon source, 1 g / L ammonium chloride (NH4CI) as the nitrogen source, and 0.5 g / L sodium chloride (NaCI) to maintain osmotic balance.

[0081] To ensure effective pH control in the presence of bacteria, the medium preferably includes a buffer, such as for example a 10 mM HEPES buffer. The pH of the minimum medium solution may be adjusted using an acid such as for example nitric acid (HNO3). Preferably, the pH adjustment ensures that the medium remains within the optimal pH range for bacterial growth, typically between 6 and 8, such as between 6 and 7.5, between 6 and 7, or more preferably between 6 and 6.5.

[0082] The culture medium can easily be chosen and adapted by the person skilled in the art. Examples of culture media include M9 minimum culture medium, Czapek-Dox minimum culture medium, etc.

[0083] Advantageously, the minimal and precisely controlled nutrient environment promotes the formation of a robust biofilm on the glass surface.

[0084] Process

[0085] The process according to the present description enables the formation of biofilms of microorganisms, in particular bacteria, on a glass matrix.

[0086] The process according to the present description is particularly suitable for extracting at least one chemical compound, in particular at least one metal, from glass. Thus, in one embodiment, the process according to the present description is implemented in a process for extracting at least one chemical compound, in particular at least one metal, from glass. Preferably, the chemical compounds extracted from glass, in particular the metal, are selected from the group consisting of manganese, iron, sodium, potassium, calcium, magnesium, and mixtures thereof.

[0087] Preferably, the extraction process according to the present description comprises at least one step of carrying out the process of growing a biofilm on a glass matrix as described in the present description.

[0088] Preferably, the extraction process is carried out for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days.

[0089] In an embodiment, the extraction process can also include a glass recovery step. Advantageously, the glass can be reused in various industrial applications.

[0090] In an embodiment, the extraction process can also include a step for recovering the extracted chemical compound, in particular the metal. Advantageously, the extracted chemical compound, in particular the metal, can be recovered and reused in various industrial applications.

[0091] The process described herein is also particularly suitable for glass recycling. Thus, in one embodiment, the process according to the present description is implemented in a process for recycling a glass.

[0092] The process of recycling a glass preferably, comprises the following steps:

[0093] - Carrying out the process of growing a biofilm on a glass matrix as described in the present description;

[0094] - Recovery of the glass;

[0095] - Recycling the glass.

[0096] Preferably, glass recovery refers to the process of separating and retrieving glass fragments or glass matrix from the biofilm and microbial suspension. Recovery of the glass after the extraction process or the recycling process can be performed by separating the glass fragments from the biofilm and microbial suspension. Any method well known to the person skilled in art can be used, including mechanical separation techniques such as filtration, centrifugation, decantation etc.

[0097] In addition, post-treatment steps can be performed to clean or sterilize the glass fragments for further use, such as rinsing with solvents, acid washes, or autoclaving.

[0098] The processes according to the present description may further comprise at least one preliminary step selected from the group consisting of:

[0099] - Collecting and sorting the glass or glass fragments; - Cleaning the glass or glass fragments, in particular to remove contaminants such as paper, plastic and metals;

[0100] - Reducing the size of the glass fragments, for example by grinding. The invention will be further described by the following non-limiting Examples.

[0101] ures

[0102] Fiqure 1 Figure 1 shows Al, Co, Fe, K, Mg, Mn, P and Si concentrations (ppb) measured by ICP-OES for glasses (a) VM0F0, (b) VM2F0, (c) VM1 Fl and (d) VM0F2 in function of time (days) during reactor experiments in the presence of P. putida. Each data point represents the mean value of two duplicates and the error bars represent the standard deviation between these duplicates.

[0103] Fiqure 2 Figure 2 shows SEM (BSE) imoges of the surfoce of gloss coupons (o) VM0F0,

[0104] (b) VM2F0, (c) VM1 F1 ond (d) VM0F2 offer 1 month of weothering in the bioreoctor in the presence of P. putida.

[0105] Fiqure 3: Figure 3 shows cumulotive normolized moss losses (NL) in Al, Co Fe, K, Mg,

[0106] Mn, P ond Si for glosses (a) VM0F0, (b) VM2F0, (c) VM1 Fl and (d) VM0F2 in function of time (days) during bioreactor experiments in the presence of the mixture of bacterial strains collected from Notre-Dame stained glass windows. Each data point represents the mean value of two duplicates and the error bars represent the standard deviation between these duplicates.

[0107] Examples: the inventors studied biofilm formation on glass samples in the presence of bacteria.

[0108] 1. Material and methods

[0109] 1.1. Glass fragments

[0110] Five glasses with a silico-calco-potassic base composition comprising the oxides SiO2, AI2O3, MgO, K2O, CaO, Na2O, P2O5, and varying manganese and iron contents (see Table 1 below) were used.

[0111] The nomenclature for these glasses has been defined as follows: VMxFy, where

[0112] - V for Glass

[0113] - M for Manganese, where x is the percentage of manganese in % of MnO and is 0, 1 or 2;

[0114] - F for Iron, where y is the percentage of iron in % FeO and is 0, 1 or 2.

[0115] Table 1

[0116] Glass samples were prepared in the form of 1 mm thick coupons with a surface area of approximately 2.53.10-3m2.

[0117] 1.2. Bacterial strains

[0118] Various bacterial strains were used.

[0119] • Pure bacterial strain: Pseudomonas putida Mn-Bl ATCC 23483 (P. putida).

[0120] Before use in the experiments, the stock solution containing the Pseudomonas putida bacterial strain was stored in sterile 1 mL eppendorfs® containing 750 pL of bacteria- rich stock solution and 250 pL of 80% glycerol solution. These eppendorfs® were stored in a freezer at -20°C. • Strain mixtures: bacterial mixtures from samples taken from medieval stained glass windows in Notre-Dame Cathedral, Paris, which had been deposited in a restoration workshop.

[0121] Microbiological sampling was carried out by rubbing a sterile swab over a 1 cm by 1 cm area of the stained glass for 30 seconds.

[0122] In the laboratory, the sample was grown in an enriched culture medium consisting of 0.75 g / L (NH4)2SO4, 0.25 g / L K2HPO4, 1 .25 g / L MgSO4.7H2O, 1 .25 g / L yeast extract and 5 g / L glucose. The bacterial sample was incubated directly in contact with the glass to select bacterial strains suitable for cultivation in the laboratory and adapted to a stained-glass environment. The culture was placed at 30°C and shaken at 160 rpm for 3-4 days.

[0123] Selected bacteria were stored in sterile 1 mL Eppendorf® containing 750 L of culture and 250 L of 80% glycerol solution. These Eppendorf® were stored in a freezer at - 20°C.

[0124] The microbial population was then determined at genus level using molecular DNA sequencing methods. The majority genus identified in the Notre-Dame de Paris sample is a member of the Streptophyta family.

[0125] 1.3. Cultivation process

[0126] Fresh bacterial cultures with identical growth and feeding times and conditions were used in all experiments. For this purpose, either the pure bacterial strain (P. putida) or the bacterial sample (culture of the liquid culture stored in the freezer) were first cultured in Lysogeny Broth (LB) liquid nutrient medium, composed of 10 g / L peptone, 5 g / L yeast extract and 5 g / L NaCI. The contents of an Eppendorf® of stock bacterial solution were emptied into an Erlenmeyer flask containing 200 mL of LB nutrient medium. The culture was incubated at 30°C and shaken at 160 rpm. After 48 hours of growth in this nutrient medium, the bacterial cells were recovered by centrifuging 10 mL of bacterial solution. The resulting bacterial pellet was washed 3 times in a 9 g / L NaCI solution during the second stage. At each washing cycle, the bacterial pellet was systematically separated from the supernatant and resuspended in NaCI solution after the first 2 cycles. After the last wash cycle, the bacterial pellet was resuspended in the minimum nutrient medium (described below) in the third step. The optical density (OD) of the bacterial solution was then checked using a spectrophotometer (Shimadzu UV-1800) and adjusted (where necessary) to a value between 1 and 1.2, by dilution or reconcentration, in order to normalize the concentrations of the bacterial solutions intended for experiments.

[0127] 1.4. Culture medium

[0128] The culture medium used is a minimum nutrient medium designed to act solely as a source of C and N for the bacteria, since these two elements are not present in the composition of the glass. The source of P required for bacterial growth was designed to come solely from the glass. This minimum medium is composed of 1 g / L glucose, 1 g / L NH4CI, 0.5 g / L NaCI, 10 mM HEPES buffer. The HEPES buffer was chosen for better pH control in the presence of bacteria. The pH of the minimum medium solution was adjusted to 6.5 ± 0.1 with a few drops of HNO3.

[0129] 1.5. Protocol

[0130] The reactor consists of a 1 L borosilicate glass reactor with a PTFE lid. In the reactor, three 1 1 mm diameter coupons of the same glass composition were placed on the PTFE supports. The solution was stirred using a magnetic PTFE rod and a magnetic stirrer (Stuart UC151 ). The reactor was then filled with 1 L of minimum culture medium, pH initially adjusted to 6.5. To this volume were added 5 mL of a bacterial solution with an optical density close to 1 . The flow rate of culture medium injected into the bioreactor was maintained at 0.5 L / d using a peristaltic pump (Ismatec), and the solution was replenished using two bottles of concentrated solutions, one of glucose and one of glucose-free minimum nutrient medium. The temperature was kept constant at 30°C throughout the experiment, thanks to the glass reactor's double jacket, which allowed water maintained at 30°C to circulate through a circulating chiller (Julabo EcoChiller F250). Air was injected into the reactor via a needle, with a cellulose acetate filter (pore diameter 0.2 m) at the outlet to ensure sterility of the injected air.

[0131] Reactor experiments were carried out on four glass compositions (VM0F0, VM2F0, VM1 F1 and VM0F2) in the presence of pure P. putida bacteria and the bacterial mixture collected from the stained glass windows of Notre-Dame Cathedral in Paris. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) was used during this study to monitor the elements in solution following dissolution of the various glasses in aqueous media. Analyses were carried out on a Perkin Elmer Optima 8300 ICP-OES at the LGE. All samples were acidified to 5% v / v using a 65% v / v HNO3 solution. In addition, for each sample, the measurement is the average resulting from three successive analyses.

[0132] 2. Results of experiments with the P. putida strain

[0133] 2.1. Biofilm formation

[0134] Initially, an experiment was carried out on VM2F0 glass, for very short periods (1 or 3 days) to observe the early stages of biofilm formation on the glass surface. The samples were then subjected to a critical point bypass (CPD) drying technique (Leica EM CPD300), to preserve the bacterial cells and their exopolymer network.

[0135] Glass coupons with a biofilm layer recovered at the end of short-term bioreactor experiments were placed in various ethanol baths of increasing concentration: 0% (osmosis water), 30%, 50%, 70%, 96% and then 100%. These steps dehydrated the samples, replacing all the water in the bacterial cells with ethanol. The samples were then dried using a critical point bypass dryer (Leica EM CPD300), and immediately metallized with a carbon layer. This preparation method enabled the structures of bacterial cells and biofilms to be observed using SEM.

[0136] The glass coupons were observed using SEM. After one day, we can see a fairly significant bacterial proliferation on the surface of the glass (a count of around 50 bacteria on a surface area of around 65 m2). A network of exopolymers is already clearly visible, representing the first stage of biofilm formation. The adhesion of bacteria to the glass surface via these exopolymers is clearly visible. After 3 days, the number of bacteria on the surface of the glass is greater, indicating bacterial proliferation, their network of exopolymers has also developed considerably, and the points of attachment to the glass surface are very numerous. 2.2. Solution analysis

[0137] Concentrations of the elements AL, CA, Fe, K, Mn, Si and P in solution were measured by ICP-OES for VM0F0, VM2F0, VM1 Fl and VM0F2 glasses (see Figure 1 ).

[0138] For each glass composition, the presence of glass elements in solution is observed from the first sampling, which is carried out after half a day's experiment. Alkaline and alkaline-earth elements, in particular, are released in preference to silicon. Iron and manganese are both released in solution.

[0139] 2.3. Characterization of weathered glass coupons

[0140] The glass coupons were observed using an optical microscope at the end of the experiments and after complete drying. All fourglass coupons were covered with thick biofilms.

[0141] A brown coloration is visible after biofilm drying on VM2F0 glass. This coloration is indicative of the production of manganese oxides within the biofilm.

[0142] The top surface (not in contact with the PTFE support) of each glass coupon was observed by SEM after surface carbon deposition (see Figure 2). Once dry, the biofilm is very compact and strongly attached to the glass weathering layer. Part of the biofilm was also removed from each glass coupon so that the weathered surface underneath could be observed.

[0143] The biofilms formed are highly adherent to the surface of the glass samples, and when these were mechanically removed they tore off a large part of the glass's weathering layer.

[0144] On VM2F0 glass, part of the weathered layer was removed, and the glass layer underlying the weathered layer could be observed. SEM EDS (Energy Dispersive Spectrometry) analysis determined that this underlying glass layer contains silicon, phosphorus, aluminum, potassium and manganese. These are the main elements in the glass matrix. The glass is therefore “healthy”, having undergone very few variations in its composition. The altered glass layer, on the other hand, is composed of silicon, aluminum and a little manganese - in other words, a silica-rich phase devoid of alkalis and alkaline earths.

[0145] 3. Results of bioalteration experiments in an open aqueous medium using a mixture of bacterial strains collected from the stained glass windows of Notre-Dame de Paris

[0146] 3.1. Biofilm formation

[0147] A short-term (3-day) bioreactor experiment was carried out in the presence of a mixture of bacterial strains collected from Notre-Dame de Paris stained-glass windows, to observe bacterial attachment to the glass surface. This experiment was carried out on VM2F0 glass only. At the end of the experiment, the samples were subjected to CPD drying to preserve the bacterial cells and their exopolymer network.

[0148] After this short time, numerous bacteria are visible on the surface of the glass (around 30 bacteria over an area of around 200 pm2). Around the bacteria, a large network of exopolymers can be observed. Bacterial cells adhere to the glass surface via these exopolymers. Several bacterial cell shapes can be seen in the SEM image. Two or three bacterial shapes can be seen, providing evidence of the proper development of the mixture of bacterial strains collected in situ.

[0149] 3.2. Solution analysis

[0150] The experiments were carried out over periods of 1 month, during which the solution was analyzed to characterize glass weathering.

[0151] Concentrations obtained by ICP-OES have been normalized to cumulative NL for each glass element and are presented in Figure 3.

[0152] Normalized mass losses (NLi) were calculated with Equation 1 :

[0153] Equation 1 : NLi = (Q*Ci*t) / (xi*S) where:

[0154] Ci is the concentration of element i (g.mr3) in the sample, t is time (days), xi is the mass fraction of element i in the glass composition,

[0155] S is the reactive surface area (m2) of the glass in contact with the solution, and Q is the solution flow rote (m3 / d).

[0156] These accumulated NL are representative of glass element mass losses throughout the experiment.

[0157] For each glass composition, the presence of glass elements in solution is observed. Alkaline and alkaline-earth elements, in particular, are released preferentially to silicon. Iron and manganese are both released in solution. All glasses are altered.

[0158] 3.3. Characterization of weathered glass coupons

[0159] The four glass coupons are covered with thick biofilms.

[0160] To characterize the weathered surface of the glasses, the glass coupons were resin- coated, then sawed and polished on edge.

[0161] A cross-section of each glass shows healthy glass in the central part, surrounded on both sides by a layer of glass clearly identified as a layer of weathered glass. A third layer above the other two corresponds to the biofilm.

[0162] Ten measurements of the weathering layer and biofilm were taken on each image.

[0163] The values are summarized in Table 2 below.

[0164] Table 2 :

[0165] 4. Conclusion

[0166] The results show that bacteria were able to grow on the glass surface in all reactors, regardless of the glass composition present. In fact, all glasses are covered with a thick biofilm adhering to the glass surface, in response to the continuous conditions of the bioreactor device. The nutrients contained in the glass are sufficient to trigger bacterial growth and the production of consistent biofilms clinging to the glass surface. The results also show that biofilms formed on glass surfaces have the ability to extract compounds from the glass.

Claims

25AMENDED CLAIMS received by the International Bureau on 19 March 2026 (19.03.2026)1. A process for extracting at least one metal from glass by growing an adherent biofilm on a glass matrix, the process comprising a step of contacting glass fragments with at least one microorganism in an open flow reactor with a continuous flow of culture medium, wherein the renewal rate of the culture medium is adjusted to provide a residence time of the at least one microorganism suspended in the open flow reactor of at least two days.

2. The process according to claim 1 , wherein the at least one microorganism is at least one bacteria.

3. The process according to claim 1 or 2, wherein the at least one microorganism is a bacteria selected from the group consisting of bacteria of the genus Bacillus, Arthrobacter, Paenibacillus, Gloeocapsa, Nostoc, Streptomyces, Pseudomonas, Burkholderia, Agrobacterium, Sphingomonas, Acinetobacter, and mixtures thereof.

4. The process according to any one of claims 1 to 3, wherein the glass fragments comprise at least one oxide selected from the group consisting of SiO MnO, FeO, SIO2, AL2O3, MgO, CaO, Na2O, K2O, B2O3, TIO2, ZnO, CuO, CU2O, NiO, U2O, CoO, La2Os, ZrC>2, PbO, P2O5, and mixtures thereof.

5. The process according to any one of claims 1 to 4, wherein the temperature in the open flow reactor is comprised between 20°C and 40°C.

6. The process according to any one of claims 1 to 5, wherein a culture medium which is a minimal nutrient medium is introduced in the open flow reactor.

7. The process according to any one of claims 1 to 6, wherein the pH of the culture medium introduced in the open flow reactor is between 6 and 8.

8. The process according to any one of claims 1 to 7, wherein the process is carried out in the presence of an excess of oxygen.

9. The process according to any one of claims 1 to 8, wherein the at least one metal is selected from the group consisting of manganese, iron, sodium, potassium, calcium, magnesium, aluminum, and mixtures thereof.

10. The process according to any one of claims 1 to 9, wherein the process is carried out for a period of at least 2 days.

11. A process according to any one of claims 1 to 10, further comprising a step of recovery of the glass.

12. A glass obtained by the process as defined in any one of claims 1 to 1 1 .

13. A process of recycling a glass comprising the following steps:- Carrying out the process as defined in any one of claims 1 to 1 1 ;- Recovery of the glass;- Recycling the glass.