Process for the bio-cleaning of the surfaces of objects of various chemical natures and buildings

The present invention relates generally to a process for the cleaning of the surfaces of objects, of various chemical natures, and buildings. Particularly, the invention refers to a biocleaning process of said surfaces through the use of microorganisms .

In outdoor environments in recent decades, particularly in urban areas, surface alterations such as nitration, sulphation, black crust and deposits of various kinds on the surfaces of buildings and objects of various chemical natures have increased significantly. Particular and frequently occurring cases are the- changes caused by atmospheric pollutants such as nitrogen oxides and sulphur dioxides. In air, the above-mentioned compounds are oxidised to nitric acid and sulphuric acid which, following deposition on surfaces, are in turn converted into nitrates and sulphates. The formation of nitric acid (HNO ₃ ) and sulphuric acid (H ₂ SO ₄ ) , and their subsequent deposition on surfaces of various chemical natures, such as for example stone or metallic surfaces, in the form of acid rain, causes the transformation of

the substrate and consequent nitration and sulphation. In addition, the aforementioned black crusts are the result of the action of sulphuric acid on calcium carbonate (CaCO ₃ + H ₂ SO ₄ → CaSO ₄ .2H ₂ O + CO ₂ ) with the consequent formation of calcium sulphate, or gypsum, which traps carbonaceous (carbon-containing) residues. Nitration and sulphation phenomena may be caused, not only by pollution, but also salt movement processes, for example due to rising up from the soil .

In confined spaces, such alterations can be caused by agents other than atmospheric pollutants. Particularly , previous restoration work, based on the application of organic and inorganic substances, may over time be considered alterations in themselves.

In order to treat the surfaces of buildings and objects of various chemical natures, chemical products are normally used, often in combination with mechanical action. Although in certain cases chemical action, optionally combined with mechanical action, has been shown to be effective in the removal of alterations, certain significant drawbacks have been observed.

First of all, cleaning operations using chemical substances are normally performed using organic solvents and solubilising agents which can give unsatisfactory results, be too aggressive in relation to the substrate,

be poorly selective, pollute the environment and can be hazardous for the operators and potentially cause changes in the optical properties of the substrate itself.

Mechanical action involves particularly delicate and precise execution, so as to avoid irreparable physical damage to the surface beneath the alteration, and hence sometimes requires long periods of time.

In order to remove surface alterations such as nitrification, sulphation and black crust, the use of denitrifying and desulphurising microorganisms applied to the surfaces to be treated has been proposed, in order to obtain biological type cleaning, on the one hand avoiding the use of overly aggressive substances and methods and the handling of potentially toxic substances by cleaning staff, while on the other hand accelerating cleaning times .

The application of microorganisms has been tested for several years, and has been shown to be a valid alterative to the above-mentioned systems. Particularly , it has been proposed to treat marble statues by immersion in a bath of desulphurising bacteria in order to eliminate the incrustations caused by hydrated gypsum (Gauri et al., 1989, The sulfation of marble and the treatment of gypsum crusts. Stud. Cons. 34:201-206).

However, said application implies drawbacks

associated with the volume of the treatment bath with respect to the dimensions, generally significant, of the objects to be treated. For example, treatment of this kind cannot be used for buildings. Furthermore, a consolidation step of the statue is required prior to treatment in order to avoid serious damage caused by immersion in the treatment bath.

Alternatively, the use of sepiolite has been proposed as a support medium for trapping the microorganisms in a matrix and applying said medium to the object to be treated (Ranalli et al. , 1997, The use of microorganisms for the removal of sulphates on artistic stoneworks, Int. Biodet. Biodegr. 40:255-261) .

This method requires long and laborious preparation (at least one week to allow microbial growth and colonisation of the sepiolite particles) . Furthermore, the system does not maintain favourable conditions, particularly water availability, so that the microorganisms can carry out their action efficiently and so a large concentration of microorganisms is required in order to overcome the adverse conditions.

Organic supports, such as polyacrylic acid, have been tested in order to obviate the problems associated with the colonisation of inorganic supports. The microorganisms are entrapped in the organic gel during

gel formation, which requires ten minutes or so on average. On the contrary, in the case of inorganic supports, colonisation generally requires growth and adhesion onto the particles of the inorganic matrix, a process requiring 7 days at least .

It is also true that organic supports, such as the polyacrylic acid mentioned, have a great limitation represented by the very poor capacity to adhere to any type of stone (e.g. both smooth and rough plaster) or metallic items etc.

It should also be borne in mind that the presence of microorganisms causes the release of catabolytes, including certain substances such as for example organic and inorganic acids, which are in turn corrosive. In the case of desulphurising bacteria, iron sulphide precipitates form in the culture medium in which the microorganisms are made to grow prior to entrapment in the support medium.

The technical drawback underlying the present invention is that of providing a process for the cleaning of objects of various chemical natures and buildings, involving the use of microorganisms for simple and effective cleaning.

Said problem is resolved by the use of a support medium for microorganisms, capable of maintaining the

necessary and sufficient conditions, so that they can fulfil their role as biocleaners of variously altered objects of various chemical natures and buildings.

A first object of the invention is hence that of providing a more appropriate support for microorganisms that can be applied to the surfaces of objects and buildings to be treated.

A second object is that of providing a process for cleaning the surfaces of objects and buildings comprising the use of said support .

A further object is that of providing a kit for the application of said process .

Further characteristics and the advantages of the present invention will be more evident from the following detailed description of an embodiment, given purely by way of non-limiting example.

The idea underlying the invention is that of devising a system for supporting a sufficient number of the cells of interest so as, on the one hand, to guarantee minimum viability, necessary for fulfilling their role as biocleaners, and on the other hand, optimal adhesion to any type of object or building with alterations of various kinds .

Following numerous experiments, an optimal support for biocleaning microorganisms has been developed which

mainly satisfies the aforementioned desired requirements. Said support (which, if together with microorganisms, is referred to hereinafter as biological formulation) is in the form of a matrix comprising an organic polymer compound in which a silicate-based inorganic compound or a silica is dispersed.

In numerous experiments, polymer compounds have been tested, generally based on polymers forming a lattice capable of entrapping the cells of the microorganisms. Preferably, the compound is in the form of an organic hydrogel. For example, the polymeric compound may be a mixture of functionalised acrylamides (PAAM) with different molecular weights or a polyvinyl alcohol (polyacrylamide, polyvinyl alcohol and water) . Particularly , the polymer is an acrylic resin hydrogel such as Hydrobiogel 97 (EniTecnologie, San Donato, . Milan) .

Preferably, the polymer is acrylic resin based, capable of forming lattices and entrapping microorganisms. More preferably, said compounds, in the form of acrylic polymers, are polyacrylic acid (propenoic acid monomer) Carbopol ^® 934 (CST, Vicenza) , this is a hydrophilic polymer lattice which swells in water and, by not dissolving, begins to disperse due to the effect of the high M.W. of the molecules and a pH of approx. 3.0,

and appears to have low viscosity; the modified polyacrylic acid Carbogel (CST, Vicenza) , obtained through the basic alkalinisation of Carbopol ^® 934. The preparation of the modified polyacrylic acid gel (Carbogel) occurs through the simple addition of water, and the viscosity of the gel can be modified by varying the quantity of polymer added to a given volume of water. Particularly , the formation of a Carbogel can include a quantity by weight of organic compound in a volume of water of between 0.1% and 15.0%, depending on the need for greater or lesser gel fluidity, preferably between 1.0% and 4.0%.

In general, the silicate or silica based inorganic compound can be in any compound, which when mixed with the organic polymer matrix, alters the thixotropic properties, making it suitable for application to any type of surface. Particularly , the inorganic compound may be any silicate-containing compound such as, for example, type T-O (1:1) and type T-O-T (2:1) phyllosilicates, such as sepiolite and silica. Preferably, said compound is represented by a hydrated silica such as micronised silica. More preferably, the inorganic compound is hydrated micronised silica, manufactured by PPG Industries (Pittsburgh, PA) under the brand name Lo-Vel ^® 27 (CAS Registry No. 63231-67-4) .

Particularly , this product belongs to the family of amorphous synthetic silicas, and appears in the form of a dry white powder (particle size of 2 microns) with specific surface area of 180 m ² per gram of product.

It has been surprisingly found that a support such as that described, constituted by organic and inorganic components, possesses unexpected properties.

First of all, thanks to the action of the organic polymer, it is capable of entrapping an adequate number of microorganisms and keeping them alive for a useful period of time so that they can perform their biocleaning role, while maintaining the necessary water content for achieving the desired microbial activity.

Secondly, thanks to the action of the inorganic component, for example the above-mentioned micronised silica, the support may be easily applied onto both horizontal and vertical surfaces or ceilings without encountering any undesired detachment. Indeed, it has been observed that polymer matrices alone, when applied to walls/ceilings, may become easily detached, above all if the walls are vertical or vaulted, as occurs frequently for example with historical monuments.

Furthermore, it has been observed that the preparation of the support with microorganisms is significantly accelerated with respect to the use of the

inorganic component alone. Indeed, the preparation time of the polymer matrix with the entrapped microorganisms is only around 10 minutes, compared with the above- mentioned at least 2 days necessary for the colonisation of certain inorganic supports such as sepiolite.

Particularly , for 100 mL of suspension, the support of the invention may comprise between 0.1 and 15 g by weight, preferably between 1 and 4 g (for example Carbogel) and between 5 g and 20 g, preferably between 8 g and 15 g of inorganic compound (for example micronised silica) . The ratio between the two organic and inorganic components may vary in relation to the efficacy and efficiency of adhesion and anchorage of the final formulation with respect to the chemical nature of the material to be treated, the recumbent position of the object itself (vertical, horizontal, inclined, vaulted, etc.), the weather conditions (high or low temperature, humidity, etc.) and the oxic (in air, in the presence of oxygen), or anoxic (in the absence of oxygen) conditions.

When microorganisms are added to the substrate, a biological formulation is obtained in which different microorganisms such as prokaryotes, for example bacteria, and eukaryotes for example yeasts, microfungi in general, are entrapped in an organic and inorganic matrix, as described previously.

By way of example, the formulation may include desulphurising and/or denitrifying bacteria capable of removing sulphates and nitrates respectively.

The desulphurising bacteria may be selected from the genuses Desulfovibrio, particularly from the species Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Among the Desulfovibrio vulgaris bacteria, the preferred bacterium is Desulfovibrio vulgaris subspecies vulgaris, more preferably, it is the bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579. Among the Desulfovibrio desulfuricans bacteria, the preferred species are Desulfovibrio desulfuricans ATCC 13541 and ATCC 29577. In general, all the microaerotolerant and aerotolerant desulphurising bacteria may be used.

The denitrifying bacteria may be selected from the genus Pseudomonas, particularly Pseudomonas stutzeri. Preferably, the species are Pseudomonas stutzeri strain A29 DISTAM, Pseudomonas stutzeri DSMZ 5190, Pseudomonas stutzeri ATCC 23856.

The biological support of the invention may be prepared in accordance with a process comprising the preparation of an aqueous solution wherein, in every 100 mL of water are dissolved by stirring between 0.1 g and 15 g, preferably between 1 and 4 g per 100 mL, of the polymer component reported above, and between 5 g and

20 g, preferably between 8 g and 15 g, of the inorganic component .

Once the support is prepared, the microorganism of interest can be added in the form of a cell suspension so as to give the formulation, ready for the application of interest .

Alternatively, the support of the invention may be prepared directly in a cell suspension, so as to create the polymer matrix capable of supporting the microorganisms within said cell suspension. The step of adding the organic compound to the cell suspension generally occurs slowly, in the above-reported quantities, and with stirring for 7-10 minutes.

In this case, the preparation of the organic component normally comprises the addition, to 100 mL of cell suspension, of 0.1-10 g of polymer and stirring so as to obtain a gel under oxic conditions (in air) , while under anoxic conditions (in the absence of oxygen) approx. 1-15 g are added (while maintaining gentle stirring of the mass using a mechanical stirrer at 50 rpm) .

Subsequently, between 5 and 15 g of the inorganic component, such as for example micronised silica, is added to the cell suspension with organic polymer.

The cell suspension is prepared in accordance with

normal microbiological techniques known in the sector, i.e. growing the microorganisms in a suitable culture medium until reaching the desired cell biomass. In the case where strains derived from national and international microorganism collections are used, such as those reported above, the preparation conditions are essentially those recommended by the collections themselves .

A further innovative element and motive of the present invention is in specifying the identification of a process capable of resolving the undesired phenomena described and the problems associated with the preparation and use of microorganisms. Particularly , when desulphurising microorganisms are used, these form hydrogen sulphurate, which, in the presence of ferrous ions in the conventional culture media adapted for such organisms, causes the formation of a black inorganic precipitate (iron sulphide) .

Indeed, in order to overcome this drawback, the cell suspension preparation process comprises a step of transferring the cell culture, grown in conventional medium, into a medium free of iron ions and its incubation for approx. 2-4 days under anoxic conditions.

Preferably, the preparation also comprises a subsequent step of eliminating inorganic precipitates,

preferably by means of filtration using filters with minimum pore diameters comprised of between 2 and 10 μm, preferably 8 μm, which retain any precipitate and allow the cells to pass through.

It has been observed that the precipitate filtration step normally used is very slow and troublesome, as the filters have narrow filtration surfaces and tend to become clogged quickly.

In order to speed up the filtration step, a pre- filtration step has been advantageously introduced with glass wool filters, with the scope of separating as much precipitate as possible and thus aiding the proper filtration step.

Afterwards, the cells are recovered and resuspended in a suitable buffer, such as potassium or sodium

I phosphate, prior to being added to the organic polymer component, or to the mixture of said component with the inorganic component .

However, it has been found that this latter step of resuspension in potassium/sodium phosphate buffer can in reality also cause the drawback of the release of potassium/sodium ions, which remain on the surface of the object, and are themselves sources of alteration.

In order to resolve this further problem, the potassium/sodium phosphate has been replaced with

ammonium phosphate, which releases the ammonium in the form of ammonia. Being a gas, ammonia is released into the air without depositing salts on the object being treated. However, in general, all non-toxic buffers which are effective around neutral pH and do not inhibit the activity of the microorganisms can be used.

For anaerobic microorganisms, all the cell suspension preparation steps described take place under anoxic conditions .

The support of the invention, which can be defined biological formulation, is now ready to be applied onto the surfaces of objects or buildings to be treated with biocleaning microorganisms, in accordance with the procedure reported hereinafter.

The biocleaning method of the invention comprises the steps of: providing a biological formulation like that described previously; applying a layer of said formulation onto a surface to be treated; leaving said layer on the surface for a sufficient period of time for the microorganisms to perform their biocleaning action; removing said layer once their action has

terminated .

Particularly , the biological formulation layer application step may be achieved through the use of a spatula. The layer is applied uniformly, with a thickness comprised of between 0.5-3 cm.

Furthermore, in order to enhance the retention of the water contained in the organic polymer, and reduce the diffusion of oxygen, in the case of anaerobic microorganisms, the biological formulation is in turn covered with an elastic plastic film. This way, the action of anaerobic microorganisms can be enhanced.

Following application, the biological formulation is left in position for a period of time varying between 6 hours and 7 days, more preferably between 8 and 72 hours, depending on the type of microorganism or the conditions of the chemical composition of the object to be treated. Several successive applications can be envisaged.

If the environmental conditions (e.g. the temperature, and in the case of rain) are not favourable, simple contrivances, all within the ability of one skilled in the art, can be adopted. For example, the operating temperature of the microorganisms may be adjusted by the application of a black plastic film, optionally in association with a heat lamp, in order to balance out any drops in temperature. For more arid

climates, it is possible to increase the number of applications so as to apply a fresh layer of formulation and so hydrate and vary the quantity of organic component. Furthermore, in the case of open air objects, contrivances may be adopted in order to avoid washing away due to rain and other undesired events (wind, snow etc.) such as for example resorting to fixed covers (roofs, portable or fixed shelters, etc.).

Preferably, application also comprises a preliminary step of overglazing the surface to be treated with Japanese paper (with grammage of 0.20-0.40 g/m ² ) soaked in distilled water (or the same buffer) in order to aid the step of removing the layer of formulation on completion of the biocleaning treatment.

Once the action of the biocleaning organisms is complete, the biological formulation is removed, for example, by means of removal of the Japanese paper.

As mentioned previously, the main advantage of the present invention resides in the fact that the support has been conceived in such a way as to guarantee optimal conditions for the survival of the microorganisms on the one hand, and on the other hand, improved adhesion onto the surfaces to be treated without, moreover, interfering with the activity of the microorganisms themselves.

Indeed, thanks to the particular combination of the

organic polymer component with the inorganic component, the microorganisms have a sufficient quantity of water for survival, with a limited extent of loss of cell vitality over time, and, at the same time, can be applied to any type of surface, even in vertical positions/ceilings such as the arches of monuments or buildings in general, without becoming detached due to the weight of the polymer component and its poor adhesion to said surfaces when used without the aforementioned inorganic component.

Furthermore, the process, aimed at developing the optimal formulation, has been created by devising ideal preparation conditions, so that the microorganisms entrapped therein can act without giving rise to undesired side effects themselves.

As reported previously, the microorganisms are prepared in such a away that their subsequent entrapment in the polymer component occurs under the best conditions, in other words by eliminating or at least significantly reducing the formation/production and release of undesired substances on the surface of the substrate being treated.

A further advantage arises from the fact that the preparation of the biological formulation, starting from a cell suspension, its application and function, have

been significantly accelerated allowing correspondingly significant time savings, preparing everything in just a few minutes with respect to the 7 days and more in the case of the use of colonised sepiolite as matrix.

A further object of the invention is a kit for biocleaning the surfaces of objects of various chemical natures and buildings, comprising: an organic component, as described previously; an inorganic component, as described previously; an optional buffer or medium; biocleaning microorganisms of interest, such as those described previously; means of applying biocleaning microorganisms to a surface to be treated; an explanatory leaflet containing instructions for the use of the kit.

Particularly , the organic and inorganic components, the buffer/medium and the microorganisms are contained in specific standard containers such as sealed bottles or bags.

Alternatively, the organic and inorganic components may be pre-mixed and ready to be combined with the microorganisms immediately prior to use.

The microorganisms may be stored living in lyophilised form and hydrated at the time of use by the buffer/medium, which is preferably an aqueous solution capable of restoring the viability of the microorganisms, and at the same time form the gel matrix of the organic and inorganic component of the biological application to be applied.

The means of application may be represented by conventional spatulas, modified brushes or other tools known in the sector.

Some embodiments of the invention, given purely by way of non-limiting example, will be reported hereinafter. EXAMPLE 1

A process for the preparation of the bacterial microorganisms Desulfovibrio vulgaris subsp. vulgaris ATCC 29579

The preparation of the cell suspension comprises the use of the preferred microorganism Desulfovibrio vulgaris subsp. vulgaris ATCC 29579.

The strain ATCC 29579 is maintained in Desulfovibrio DSMZ 63 medium (0.5 gl ^"1 K ₂ HPO ₄ , 1.0 gl ^"1 NH ₄ Cl, 1.0 gl ^"1 Na ₂ SO ₄ , 0.1 gl ^"1 CaCl ₂ x2 H ₂ O, 2.0 gl ^"1 MgS0 ₄ x7 H ₂ O ₇ 2.0 gl ^"1 DL-Na-lactate, 1.0 gl ^"1 yeast extract, 1.0 mgl ^"1 resazzurina, 0.5 gl ^"1 FeS0 ₄ x7 H ₂ O, 0.1 gl ^"1 Na-

thioglycolate, 0.1 gl ^"1 ascorbic acid) and incubated at 30 ⁰ C for 4 days under anoxic conditions.

For the removal of any sulphides generated, prior to everything, modified DSMZ 63 medium, which does not contain any sources of iron is used (unlike standard DSMZ 63 it does not contain FeSO ₄ x7H ₂ O) . Strain ATCC 29579 maintained in standard DSMZ63 medium, also grows efficiently in iron-free medium if the inoculum is less than 8% (v/v) . Once grown in the modified medium, a pre- filtration step is performed using glass wool, which retains the black iron sulphide precipitates and allows the cells to pass through, followed by proper filtration using a Rapida A perfecte ^® cellulose filter (Cartiera di Cordenons, Vicenza, Italy) with porosity of 8 μ allowing entrapment of the precipitates but leaving the cells to pass through.

Following filtration, the cells are washed by centrifugation (twice at 8000 rpm for approx. 10 minutes) with buffer.

At this point, the cell pellet is recovered and resuspended in ammonium phosphate buffer at pH 7 made with 1 g/L (NH ₄ )H ₂ PO ₄ and 1.1 g/L (NH ₄ J ₂ HPO ₄ supplemented with 0.599 g/L sodium lactate to give a cell density of approx. 10 ⁸ cells/mL. EXAMPLE 2

A process for the preparation of the formulation with sulphate reducers and application thereof

Once a cell suspension as described in example 1 is prepared, I g of Carbogel is mixed in 100 mL of suspension under anoxic conditions, until a dense suspension is obtained, followed by the addition of 13 g of micronised silica (Lo-Vel ^® ) . The area to be treated, altered by black crust, is covered with Japanese paper soaked in distilled water so as to obtain perfect contact with the surface of the altered stonework. The biological formulation is applied using a spatula until a thickness of approx. 1 cm is obtained, which is finally covered with plastic film. The overall duration of the application is 45 hours, with three successive applications of 15 hours each. Removal of the sulphates is assessed by ion exchange chromatography, which shows 98% removal of the sulphates. EXAMPLE 3

A process for the preparation of the biological formulation for the removal of organic substances (oxic conditions)

0.5 g of Carbogel are mixed in 100 mL of cell suspension under oxic conditions to give a dense suspension, and then 13 g of micronised silica (Lo-Vel ^® ) added.