IT202000016549A1 - METHOD FOR INCREASING THE CONCENTRATION OF CALCIUM CARBONATE IN A GEOMATERIAL - Google Patents

Description

DESCRIPTION

of a PATENT application FOR INDUSTRIAL INVENTION with the following title: "Method for increasing the concentration of calcium carbonate in a geomaterial".

FIELD OF THE INVENTION

The present invention relates to the field of bacteria which use urea for the formation of biocement and biomortar. The bacteria used in this invention are indigenous ureolytic bacteria isolated from the local environment, grown and scaled under non-sterile conditions. This invention? applicable to the stabilization of loose sand in deserts and for construction, such as for the strengthening of bridges, buildings and roads.

STATE OF ART

A quarter of the world's land mass, which is home to more of one billion people in 100 countries, ? threatened by desertification. In particular, the capital of Mauritania, Nouakchott, ? on the verge of succumbing to this threat, and in Nigeria the desert is moving southward at 600m/year. Conventional soil solidification measures based on concrete and chemicals are not applicable mainly for economic reasons. Stimulating and exploiting a natural process inherent in living soil, such as a bio-mediated calcification process, such as biocementation, could make the dream of slowing down desertification come true.

In nature, three groups of microorganisms cause the precipitation of calcium carbonate, whereby biocementation occurs as a useful byproduct in them: (1) photosynthetic microorganisms such as cyanobacteria and algae which carry out photosynthesis in water and can stabilize CO2; (2) sulfate-reducing microorganisms responsible for the multistage dissimilatory reduction of sulfate; (3) organisms that have a role in the nitrogen cycle through amino acid ammonification / nitrate reduction / urea hydrolysis. Urease-producing bacteria can be divided into two distinct groups based on the response of their urease to ammonium: a) those whose activity? urease isn't it? repressed by the presence of ammonium inside the cell and b) those whose activity? urease? repressed by ammonium, which prevents further hydrolysis of urea. because high concentrations of urea are hydrolysed in the biocementation process, only that microorganism whose activity? urease isn't it? repressed by ammonium ? useful in biocementation. Furthermore, an ideal microbial source of urease for biocementation must be tolerant of high concentrations of urea and calcium. The organism should also have a high level of activity? of urease is produced in a constitutive way, for example a quantity? enzyme constant ? expressed per cell, or pu? be induced reliably. In addition to meeting the need of biocementation, the organism must also meet the need for safe environmental application. In order to safely release an organism into the environment, it must be non-pathogenic, non-genetically modified and must not contain any transferable elements that could increase pathogenicity. of environmental strains (e.g. antibiotic resistance).

In the last few years ? A relatively green and sustainable soil improvement technique has been introduced which is called microbiologically induced calcite precipitation (MICP). This technique uses biochemical processes to improve the properties? soil engineering, such as strength and permeability. As a cross-science research field, MICP lies at the intersection of microbiology, geochemistry, and civil engineering, and plays a role in related applications, including wastewater treatment and limescale restoration.

Many studies have been conducted so far to investigate calcium carbonate precipitation via MICP in relation to soil reinforcement, surface reconstruction, bricks, conventional concrete self-healing and reconstruction of historical monuments, see for example the review by Murtala Umar et al., Journal of Rock Mechanics and Geotechnical Engineering (2016), 8, 767-774.

In MICP, urease catalyzes the hydrolysis of urea producing ammonium and carbonate ions; the production of ammonium in turn leads to an increase in pH; self ? If a source of calcium is available, the carbonate ions precipitate as calcium carbonate. The hydrolysis of urea ? the result of metabolic processes that depend on the bacteria used for this purpose. Many microorganisms naturally present in the soil, in particular the bacteria of the Bacillacae family, are able to hydrolyse urea (NH2)2CO in the presence of water into ammonium and carbonate ions by means of the following reaction

(NH2)2CO+2H2O? 2NH4<+ >+ CO3<2>

The generation of NH4<+ > ions increases the local pH and, in the presence of calcium ions and the availability? of nucleation sites, carbonate ions spontaneously react with calcium ions to form calcium carbonate by the following reaction

Ca<2+ >+ CO3<2->? CaCO3

The cytoplasm of Sporosarcina pasteurii contains urease, this bacterium is found in abundance in soil and decomposing materials and is ? among aerobic bacteria that do not pose a threat to the environment; therefore, Sporosarcina pasteurii ? one of the bacteria used for biocementation. S. pasteurii uses an interesting method in the hydrolysis of urea that occurs together with the production of ATP, where 1) urea diffuses into the cell according to the concentration gradient; 2) urea is hydrolysed by urease which causes the alkalization of the cytoplasm to pH 8.4 and a decrease in the ?pH (difference in pH between inside and outside the cell); 3) ammonium ions are removed from the cell according to the ammonium concentration gradient, which results in an increase in the membrane potential (charge difference between the inside and outside of the cell); 4) the increase in membrane potential is reversed driving protons against the concentration gradient in the cell, which results in the generation of ATP. ? It is interesting to note that the optimal pH for the growth of S. pasteurii, for example pH 9.25, is also the pKa, met? of the dissociation constant, of the NH3/NH4<+ >equilibrium where NH3 and NH4<+ >exist in equal proportion.

, U.S. Patent Application Ser. No. 2008/0245272 discloses a method of forming a cement by combining a starting material such as rock, sand, earth or clay with amounts efficacy of (i) an exogenous urease-producing microorganism; (ii) urea, and (iii) calcium ions. A disadvantage of the Kucharski method? that the formation of calcite occurs rapidly at the injection site of the aforementioned components in the starting material, thus determining? an uneven distribution of calcite in the starting material.

The use of an exogenous urease-producing microorganism, Sprorosarcina pasteurii (DSMZ 33) in a method for precipitating calcium carbonate in soil? also discussed by Whiffin V. S. et al., Geomicrobiology Journal (2007), 24, 417-423. A five meter column of sand? been injected with bacteria, urea and calcium chloride. The precipitated calcium carbonate? been detected along the entire length of the column; however, the calcium carbonate profile was not homogeneous over the length of the column.

In order to overcome the disadvantages of Kucharski E. S. et al., and Whiffin V. S. et al., Crawford R. L. et al., US8420362 disclose a method for increasing the concentration of calcium carbonate in a geomaterial using microorganisms capable of hydrolyzing the urea to ammonia that are present in all geomaterials, the cio? indigenous microorganisms. The method involves adding a source of nutrients, such as urea and calcium ions, to the geomaterial.

SUMMARY OF THE INVENTION

Current inventors have addressed the problem of consolidation of porous materials, such as sand consolidation.

The inventors of the present have studied microbiologically mediated processes in order to produce cohesive blocks of sand with good properties? of permeability? and resistance. In particular, the inventors of the present have concentrated their studies on the microbiologically induced calcite precipitation process (MICP) in order to optimize this process both to make it less impacting on the environment and to be more? efficient and economically viable.

In this regard, the inventors of the present invention have developed a method for selectively enriching highly urease-positive bacteria from the local environment.

The method of the invention uses indigenous ureolytic bacteria isolated from the environment, see example 1, so ? safe for the environment to which the bacteria will be applied.

How will you see? in example 2 of the present application, among the ureolytic bacteria isolated from desert habitats of Iran, ten strains were identified which have an activity urease higher than that of urease purified by the corresponding standard body. Among them, Sporosarcina pasteurii MK strain accession number KT862894 showed the highest activity urease with respect to the activity? of urease purified from the standard organism Sporosarcina pasteurii ATCC 11859.

The activity? of urease, which directly controls the hydrolysis rate of urea and therefore the production of carbonate, ? the main factor controlling the formation of biocement. The activity? of urease also directly controls the pH of the environment depending on the concentration of urea, thus influencing the the production of calcium carbonate. Uncontrolled hydrolysis of urea causes the pH to rise, so when a bacterial suspension is added to the calcium salt containing the soil, calcium carbonate is rapidly produced and precipitates on the surface layer of the soil, making this layer impermeable.

Analysis of calcium carbonate production by Sporosarcina pasteurii MK strain accession number KT862894 and Sporosarcina pasteurii standard ATCC 11859 shows that the strain isolated from desert habitats produces a high level of calcium carbonate, comparable to the standard bacterium, see example 3.

The study of the influence of urea concentration on the growth of Sporosarcina pasteurii MK strain accession number KT862894 and Sporosarcina pasteurii standard strain ATCC 11859 shows that the growth of indigenous ureolytic bacteria isolated from desert habitats ? independent of the urea concentration, while the growth of the standard strain ATCC 11859 depends on the urea concentration, see example 4.

Accordingly, in the method of the invention, indigenous ureolytic bacteria isolated from the desert habitats of Iran were grown on culture media with low or moderate urea concentration, which do not promote urea hydrolysis.

The method of the present invention also comprises the homogeneous distribution and stabilization of the bacteria among the sand particles, and finally the induction of the hydrolysis of the urea.

The combination of the above phases results in the uniform distribution of the precipitated calcium carbonate in the subsurface layers.

Surprisingly, the method according to the invention overcomes the problems of the previous methods, where the production of calcium carbonate starts when the bacteria come into contact with the surface layers, which in turn results in impermeability. of the surface layer and prevention of stabilization of the subsurface layers, see example 5, 6 and 7.

Therefore, an object of the present invention ? a method for increasing the concentration of calcium carbonate in a geomaterial comprising the following steps 0) isolating indigenous ureolytic bacteria from the geomaterial;

1) cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, in which the concentration of sodium acetate is ? of 100 mM, the concentration of urea ? from 0.01 M to 0.03 M, and the concentration of protein source from 18 g/l to 22 g/l;

or

1') cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising urea and a protein source, in which the concentration of urea ? from 2 M to 3 M and the concentration of protein source? from 18 g/l to 22 g/l;

2) adding the suspension of the cultured indigenous ureolitic bacteria obtained in step 1) or 1') to a geomaterial;

3) add to the geomaterial treated in step 2) a solution comprising a calcium salt, a protein source, a bicarbonate salt, an ammonium salt and urea, and let the bacteria stabilize for a period of between 12 and 24 hours;

or

2') premixing powders of a calcium salt, a protein source, a bicarbonate salt, an ammonium and urea salt with a geomaterial;

3') add to the pre-mixed geomaterial in phase 2') the suspension of the indigenous cultured ureolitic bacteria obtained in phase 1) or 1') and let the bacteria stabilize for a period between 12 and 24 hours;

4) add to the geomaterial treated in step 3) or 3') a solution comprising a calcium salt, a protein source, a bicarbonate salt, an ammonium salt and urea, continuously for at least 2 days.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows XRD analysis of CaCO3 production by Sporosarcina pasteurii MK strain entry number KT862894.

Figure 2 shows XRD analysis of CaCO3 production by Sporosarcina pasteurii ATCC 11859.

Figure 3 shows the growth curve of the Sporosarcina pasteurii MK indigenous strain accession number KT862894 during 36 hours in the presence of 1-5 M Urea.

Figure 4 shows the growth curve of standard Sporosarcina pasteurii ATCC 11859 during 36 hours in the presence of 1-5 M Urea.

Figure 5 shows a Cartesian diagram with the results of the permeability test? to water as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Object of the present invention ? a method for increasing the concentration of calcium carbonate in a geomaterial preferably comprising the following steps 0) isolating indigenous ureolytic bacteria from the geomaterial;

1) cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, in which the concentration of sodium acetate is ? of 100 mM, the concentration of urea ? from 0.01 M to 0.03 M, and the concentration of protein source from 18 g/l to 22 g/l;

or

1') cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising urea and a protein source, in which the concentration of urea ? from 2 M to 3 M and the concentration of protein source? from 18 g/l to 22 g/l;

2) adding the suspension of the cultured indigenous ureolitic bacteria obtained in step 1) or 1') to a geomaterial;

3) add to the geomaterial treated in 2) a solution comprising from 0.01M to 0.1M of a calcium salt, from 1.5 g/l to 2.5 g/l of a protein source, from 1g/l to 2, 1 g/l of a bicarbonate salt, 5 g/l to 10 g/l of an ammonium salt and 0.01M to 0.1M urea, and allow the bacteria to settle for 12 to 24 hours hours;

or

2') premix with a powdered geomaterial of 33.2-332 g of a calcium salt, 60-200 g of a protein source, 30-60 g of a bicarbonate salt, 150-500 g of an ammonium salt and 18 -180 g of urea per 1 square meter of geomaterial in depth? of 10 cm;

3') add to the premixed geomaterial in 2') the suspension of the cultured indigenous ureolitic bacteria obtained in phase 1) or 1') and let the bacteria stabilize for a period between 12 and 24 hours;

4) add to the geomaterial treated in 3) or 3') a solution comprising from 0.1M to 1M of a calcium salt, from 1.5 g/l to 2.5 g/l of a protein source, from 1g/l to 2.1 g/l of bicarbonate salt, from 5 g/l to 10 g/l of ammonium salt and from 0.1M to 1M of urea, continuously for at least 2 days.

The indigenous ureolytic bacteria which are suitable for the method of the invention are non-pathogenic bacteria which express urease constitutively so that the urease is expressed regardless of the concentration of ammonium or nitrogen compounds. Such organisms include the following bacteria: Sporosarcina pasteurii and Sporosarcina ureae. Other microorganisms suitable for the method of the invention are non-pathogenic bacteria in which urease is expressed only in the presence of urea. An example of a bacterium in which urease is expressed only in the presence of urea? Proteus vulgaris.

Preferably, the indigenous ureolytic bacteria are selected from Bacillus fortis, Bacillus lentus, Bacillus graminis, Sporosarcina pasteurii, Sporosarcina ureae, Bhargavaeace cembensis, Virgibacillus campisalis, Escherichia fergusonii, Proteus vulgaris, and their combination. Pi? preferably, the indigenous ureolytic bacterium ? selected from Bacillus fortis strain MK accession number KT862901, Bacillus lentus strain MK accession number KT862902, Bacillus lentus strain MK accession number KT862896, Sporosarcina Pasteurii strain MK accession number KT862894, Bacillus graminis strain MK accession number KT862903, Bacillus graminis strain MK2 accession number KT862895, Bhargavaeace cembensis strain MK accession number KT862897, Virgibacillus campisalis strain MK accession number KT862898, Escherichia fergusonii strain MK accession number KT862899, Escherichia fergusonii strain MK2 accession number KT862900 and their combination.

The geomaterial used in this method can be varied provided that it has a structure with interconnected pores or fractures. The geomaterial can be rock, typically sedimentary such as a terrigenous, chemical/biochemical, or sedimentary rock. Examples of sedimentary rock suitable for the present method are conglomerate, breccia, sandstone, siltstone, shale, limestone, gypsum, dolostone and lignite. As another example, alternatively the geomaterial can? be an unconsolidated or partially consolidated porous medium such as soil (e.g. gravel, sand, silt, clay with or without organic matter such as peat) or sediment. The geomaterial of the present invention can? also be fractured igneous or metamorphic rock: even volcanic rock containing interconnected pores can? be used as the geomaterial of this method.

Can? an appropriate protein source, suitable for bacterial growth, may be required to provide energy for bacterial metabolism and reproduction.

A protein source useful for bacterial culture? the yeast extract, which ? soluble in the culture medium and does not leave suspended particles or harmful materials or residues that can affect the activity? of urease or reduce pore blockage in the geomaterial.

Other suitable protein sources are meat extract and peptone; the concentration of the protein source ? preferably 20 g/l for the bacterial culture (phases 1 and 1') and 2 g/l for the production of CaCO<3 > (phases 3 and 4).

A favorite protein source? the yeast extract, preferably the concentration of the yeast extract ? of 20 g/l for bacterial growth and 2 g/l for the production of CaCO3. According to steps 2) and 3') of the method of the invention, preferably,

- the suspension of indigenous ureolytic bacteria contains from 10<7 >to 10<12 >CFU/mL; more preferably 10<9 >CFU/mL, and/or

- the suspension of indigenous ureolytic bacteria is added to the geomaterial in quantity from 10 to 50 L/m2 with depth? of 10 cm, more preferably 30 L/m2 with depth? of 10cm.

According to step 3) of the method of the invention, preferably

- the calcium salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or - the calcium salt ? calcium chloride, more preferably ? calcium chloride;

and/or

- the protein source solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or the protein source? yeast extract, pi? preferably 2 g/l of yeast extract;

and/or

- the bicarbonate salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; and/or the bicarbonate salt? preferably baking soda, pi? preferably baking soda;

and/or

- the ammonium salt solution is added to the geomaterial in an amount from 30-100 l/m2 of geomaterial with depth? of 10 cm, more preferably 70 l/m2 of geomaterial with depth? of 10 cm and/or the ammonium salt ? preferably ammonium chloride, pi? preferably ammonium chloride;

and/or

- the urea solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10cm.

According to step 2') of the method of the invention, preferably

- powdered calcium salt is added to the geomaterial in an amount of 33.2-332 g/m² with depth? of 10 cm, preferably of 77.62 g/m2 with depth? of 10 cm; more preferably the calcium salt ? calcium chloride;

and/or

- the powdered protein source is added to the geomaterial in an amount of 60-200 g/m² with depth? of 10 cm, preferably of 140 g/m2 with depth? of 10 cm; more preferably the protein source? yeast extract;

and/or

- the bicarbonate salt powder is added to the geomaterial in a quantity of 30-60 g/m² with depth? of 10 cm, preferably 50 g/m² with depth? of 10 cm; more preferably bicarbonate salt? sodium bicarbonate;

and/or

- the powdered ammonium salt is added to the geomaterial in an amount of 150-500 g/m² with depth? of 10 cm, preferably of 350 g/m2 with depth? of 10 cm; more preferably the ammonium salt ? ammonium chloride;

and/or

- powdered urea is added to the geomaterial in an amount of 18-180 g/m² with depth? of 10 cm, preferably 42 g/m2 with depth? of 10cm.

According to step 4) of the method of the invention, preferably

- the calcium salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; preferably the calcium salt ? calcium chloride, more preferably calcium chloride; and/or

- the protein source solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or the protein source? yeast extract, pi? preferably 2 g/l of yeast extract;

and/or

- the bicarbonate salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; and/or the bicarbonate salt? preferably baking soda, pi? preferably baking soda;

and/or

- the ammonium salt solution is added to the geomaterial in an amount from 30-100 l/m2 of geomaterial with depth? of 10 cm, more preferably 70 l/m2 of geomaterial with depth? of 10 cm and/or the ammonium salt ? preferably ammonium chloride, pi? preferably ammonium chloride;

and/or

- the urea solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10cm.

According to the method of the invention, the suspension of the indigenous ureolitic bacteria added in step 2) and/or the solution of the ingredients added in step 3 and 4, are forced into the geomaterial under pressure, for example by flow or injection; or they are sprayed, dripped, or dripped onto or into the geomaterial. Alternatively, depending on the size and shape of the geomaterial, the geomaterial is immersed in them.

Preferably, according to the method of the invention, the suspension of the indigenous bacteria in step 2) is sprayed onto or into the surface of the geomaterial, preferably sand; is preferably sprayed from fixed nozzles, pi? preferably for 10-14 h, pi? preferably for 12 h and from 175 to 185 ml/h.

Preferably, according to the method of the invention, the solution of the ingredients added in step 3) is sprayed onto or into the surface of the geomaterial, preferably sand; is preferably sprayed with fixed nozzles, pi? preferably for 10-14 hours and from 175 to 185 ml/h, preferably 180 ml/h.

Preferably, according to the method of the invention, the ingredients added in step 4 are sprayed onto or into the surface of the geomaterial, preferably sand; preferably sprayed from fixed nozzles, pi? preferably for 48 to 96h, more? preferably for 72h and from 175 to 185 ml/h, preferably 180 ml/h.

According to the method of the invention, the suspension of phase 3' native ureolitic bacteria and/or the powders of phase 2' are mixed with the geomaterial.

According to the invention, a preferred method comprises

0) to isolate indigenous ureolytic bacteria from the geomaterial;

1) cultivating indigenous ureolytic bacteria, selected from Bacillus fortis strain MK accession number KT862901, Bacillus lentus strain MK accession number KT862902, Bacillus lentus strain MK accession number KT862896, Sporosarcina pasteurii strain MK accession number KT862894, Bacillus graminis strain MK accession number KT862903, Bacillus graminis strain MK2 accession number KT862895, Bhargavaeace cembensis strain MK accession number KT862897, Virgibacillus campisalis strain MK accession number KT862898, Escherichia fergusonii strain MK accession number KT862899, Escherichia fergusonii strain MK2 number of accession KT862900 and the combination thereof, preferably consisting of Sporosarcina pasteurii MK strain accession number KT862894, under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium acetate is ? of 100 mM, the concentration of urea ? less than 0.03M and more than 0.01M, and the protein source ? yeast extract 20 g/l;

2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 meter square of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm;

3') mix the pre-mixed geomaterial in 2'), preferably sand, with a suspension of said indigenous cultured bacteria obtained in step 1) and let the bacteria stabilize for a period of between 12 and 24 hours;

4) add to the geomaterial treated in 3') a solution comprising from 0.1M to 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and 0.1M and 1M urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days.

According to the invention, a more favorite includes

0) to isolate indigenous ureolytic bacteria from the geomaterial;

1) culturing the indigenous ureolytic bacterium, consisting of the strain Sporosarcina pasteurii MK accession number KT862894 under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium acetate is ? of 100 mM and the concentration of urea ? less than 0.03M and more than 0.01M, and the concentration of the yeast extract ? of 20 g/l;

2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 square meter of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm;

3') mix to the pre-mixed geomaterial in 2'), preferably sand, a suspension of said cultured indigenous bacteria obtained in phase 1) containing from 10<7> to 10<12>CFU/mL, plus? preferably 10<9 >CFU/mL, and allow the bacteria to settle for 12 to 24 hours;

4) add to the geomaterial treated in 3') a solution comprising 0.1M and 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and 0. 1M to 1M urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days.

According to the invention, a more favorite includes

0) to isolate indigenous ureolytic bacteria from the geomaterial;

1) culturing the indigenous ureolytic bacterium, consisting of the strain Sporosarcina pasteurii MK accession number KT862894 under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium acetate is ? of 100 mM and the concentration of urea ? less than 0.03M and more than 0.01M, and the concentration of the yeast extract ? of 20 g/l;

2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 meter square of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm;

3') mix to the pre-mixed geomaterial in 2'), preferably sand, a suspension of said cultured indigenous bacteria obtained in phase 1) containing from 10<7> to 10<12>CFU/mL, plus? preferably 10<9 >CFU/mL, and allow the bacteria to settle for 12 to 24 hours;

4) spraying on or in the geomaterial treated in 3') a solution comprising from 0.1M to 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and from 0.1M to 1M of urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days.

The method according to the invention preferably produces from 80 to 120 kg of calcium carbonate/m<3 > of geomaterial in 3-7 days.

EXAMPLES

Example 1

Isolation of indigenous ureolytic bacteria

Indigenous bacteria were isolated and characterized as described in Kargar M., Kargar M. Monitoring of biocement- and biogrout-producing bacteria in desert habitats of Iran. Journal of Microbial World.2018, 11:51-60.

In short, one hundred soil samples were taken at a depth of of 10 cm from several places in the deserts and sand dunes of Iran. The samples were transferred to the laboratory, cultured in a 1:10 ratio in culture media containing 5 M urea, 20 g/l yeast extract, 152 mM ammonium sulphate and 100 mM sodium acetate at pH 9, and were incubated at 30°C for 48 hours to isolate urease-positive bacteria. The culture media were prepared under non-sterile conditions, their pH values were adjusted to nine using 1M NaOH, and a culture medium ? was used as a negative control. The identification of the active strains ? was performed on the basis of conventional biochemical tests and 16S rRNA gene sequencing.

Example 2

Determination of the activity? of urease of indigenous ureolytic bacteria isolated Vs activity? of the purified urease from the corresponding standard organism.

In the absence of calcium ions, the activity? of urease ? been determined in the culture medium with a conductivity method?, since? the urease reaction involves the hydrolysis of the non-ionic substrate urea to ionic products, thus generating? a proportional increase in conductivity? under standard conditions. The rate of increase of the conductivity? (mS.min-1) ? been converted in hydrolysis of urea (mM hydrolyzed urea.min-1) by relating the measures of conductivity? of isolated samples of indigenous bacteria against standards containing purified urease from the same organism.

The isolated indigenous ureolytic bacteria were grown under standardized conditions of 1.5M urea, 25°C, pH 7.

The following indigenous bacteria isolated more? effective in the activity? of urease were identified by molecular analysis and subsequently registered with the NCBI Genbank:.

Bacillus fortis strain MK accession number KT862901

Bacillus lentus strain MK accession number KT862902

Bacillus lentus strain MK accession number KT862896

Sporosarcina pasteurii MK strain accession number KT862894

Bacillus graminis strain MK accession number KT862903

Bacillus graminis strain MK2 accession number KT862895

Bhargavaeace cembensis strain MK accession number KT862897

Virgibacillus campisalis MK strain accession number KT862898

Escherichia fergusonii strain MK accession number KT862899

Escherichia fergusonii MK2 strain accession number KT862900

Example 3

Calcium carbonate production by isolated indigenous ureolytic bacteria vs. standard ureolytic bacteria

In the presence of calcium chloride, the production of calcium carbonate crystals in the culture medium? was evaluated by X-ray diffraction (XRD) crystallography test at a temperature of 25°C and a pH of 8.5, as described in Kargar M et al cited above.

XRD analysis of CaCO3 production by Sporosarcina pasteurii MK strain accession number KT862894, one of ten indigenous bacteria isolated in example 1, and Sporosarcina pasteurii ATCC 11859 ? reported in Fig.1 and Fig.2 respectively. As shown, the isolated indigenous strain Sporosarcina pasteurii MK entry number KT862894 produced a high level of CaCO3, comparable to the level produced by the standard bacterium.

These experiments demonstrated that both isolated indigenous urea bacteria and standard urea bacteria had a high of urea hydrolyzation and thus were able to produce a high level of CaCO3.

Example 4

Influence of urea concentration on growth of isolated indigenous urealytic bacteria vs. standard urealytic bacteria in culture medium

The indigenous bacteria isolated in example 1, so? like the corresponding standard bacteria, they were grown in culture media containing 10 µM nickel chloride and 1-5 M urea. Among the isolated bacteria, Bacillus lentus MK strain accession number KT862896 showed the least growth and Sporosarcina pasteurii MK strain accession number KT862894 showed the greatest growth compared to the respective standard strain Bacillus lentus ATCC 10840 and Sporosarcina pasteurii ATCC 11859 in a 36-hour period with 3M urea.

The growth curve of the indigenous strain Sporosarcina pasteurii MK accession number KT862894 during 36 hours in the presence of 1-5 M urea are reported in Fig.3, where OD represents the density? perspective of the suspension of the Sporosarcina pasteurii MK strain accession number KT862894 Vs time.

The growth curve of Sporosarcina pasteurii standard ATCC 11859 during 36 hours in the presence of 1-5 M of Urea are shown in Fig.4, where OD represents the density perspective of the suspension of Sporosarcina pasteurii ATCC 11859 Vs time.

These experiments surprisingly demonstrated that the growth of the isolated indigenous ureolytic bacterium was independent of the urea concentration, whereas the growth of the standard bacterium depended on the urea concentration.

Example 5

Production of calcium carbonate by indigenous ureolytic bacteria isolated in the environment Example 5a

Growth of bacteria on a medium including low concentration of urea Step 1) the isolated indigenous strain of Sporosarcina pasteurii accession number MK KT862894 ? was grown under non-sterile conditions in a 100 liter fermenter containing medium comprising 0.01-0.03 M urea, 100 mM sodium acetate and 20 g/l yeast extract.

The bacteria were incubated at 30°C at 150 rpm for 24 h. What about bacterial growth? was evaluated with a spectrophotometer at 600 nm until the number of cells reached the maximum of 10<9>Cfu/mL.

Step 2') After that, a powder composition comprising 77.62 g of CaCl2, 140 g of yeast extract, 50 g of sodium bicarbonate, 350 g of ammonium chloride and 42 g of urea, ? been premixed in 1 square meter of depth? of 10 cm of sand.

Phase 3') 30 L of bacterial suspension comprising 10<9 >Cfu/mL of the bacterium grown in phase 1) ? been mixed with a square meter with depth? of 10 cm of the premixed sand obtained in phase 2'); the bacteria were allowed to stabilize for between 12 and 24 hours.

Step 4) After that, a solution comprising 0.75 M CaCl2, 2 g/l yeast extract, 1.25 g/l sodium bicarbonate and 7 g/l ammonium chloride and 0.75 M of urea ? been sprayed on the same surface of sand from fixed nozzles at 180 ml/h, continuously for at least 3 days.

Example 5b

Growth of bacteria on a medium including a moderate concentration of urea Phase 1') the isolated indigenous strain of Sporosarcina pasteurii accession number MK KT862894 ? was grown under non-sterile conditions in a 100 liter fermenter containing a medium comprising 2-3 M urea and 20 g/l yeast extract.

The bacteria were incubated at 30°C at 150 rpm for 24 hours. Bacterial growth? was evaluated with a spectrophotometer at 600 nm until the number of cells reached the maximum of 10<9>Cfu/mL.

Phase 2) A bacterial suspension comprising 10<9 >CFU/mL of the bacterium grown in phase 1'? been sprayed on a surface of sand in quantity? equal to 30 l/m2 with depth? of 10 cm from fixed nozzles at 180 ml/h for 12 hours.

Step 3) After which a solution comprising 0.05 M CaCl2, 2 g/l yeast extract, 1.25 g/l sodium bicarbonate, 7 g/l ammonium chloride and 0.05 M solution of urea ? was sprayed on the same surface of sand from fixed nozzles at 180 ml/h for 12 hours, the bacteria were allowed to stabilize for a period between 12 and 24 hours.

Step 4) After that, a solution comprising 0.75 M CaCl2, 2 g/l yeast extract, 1.25 g/l sodium bicarbonate, 7 g/l ammonium chloride and 0.75 M urea ? been sprayed on the same surface of sand from fixed nozzles at 180 ml/h, continuously for at least 3 days.

Both embodiments 5a and 5b) of the method according to the invention produce 100 kg of calcium carbonate/m<3 > of soil in 24 hours.

In both embodiments 5a and 5b) of the method according to the invention, the bacteria, grown with a low or moderate urea concentration which does not favor urea hydrolysis, were homogeneously distributed among the sand particles and stabilized, then in phase 4 they were induced to initiate the hydrolysis of urea.

The combination of the above steps resulted in the uniform distribution of the precipitated calcium carbonate in the subsurface layers.

Therefore the method according to the invention surprisingly overcomes the problems of the previous methods, in which the production of calcium carbonate starts when the bacteria come into contact with the surface layers, which in turn results in the impermeability of the material. surface layer and in preventing stabilization of subsurface layers. Example 6

Permeability test? to water in a stabilized environment

The infiltration of water into the ground? been measured through double loops. In this test, the ground? been exposed to two iron rings with a height of 30 cm, a ring more? large of 30 cm in diameter and one more? small 20 cm in diameter. The rings were propelled by a sled club a third of the way up the ground. Between the two rings? water was poured to a height h. After resetting the time, the level of infiltration ? been measured in an instant. The h and t values were recorded at different times. The water ? been stored in the pores formed on the surface of the soil up to a depth? of fifteen centimeters for a longer period? long, as shown in Figure 5.

Example 7

Compressive strength in a stabilized environment

The compressive strength test? was conducted through ASTM on 300?150 mm (12?6 inch) cylindrical specimens. ? It is essential to impregnate the walls of the mold with a thin layer of mineral oil to avoid adhesion of the sand samples after stabilization. The compressive strength of each sample ? was determined in the press at a given time by dividing the maximum force recorded by the cross-sectional area.

Time 14 days

Strength at 1600KPa (1.6MPa)

compression

Under laboratory conditions, strengths, for example the unconfined compressive strength (UCS), have been achieved from 300kPa up to 30MPa. In most cases, ? a low resistance up to 1500kPa was sufficient; in some specific cases, such as the prevention of liquefaction, ? Only a minor increase in strength up to 150kPa was needed to prevent sand creep. The growth of the isolated indigenous bacteria used in the method according to the invention was not necessarily dependent on urea, as it is? seen in example 4.

In both embodiments 5a and 5b) of the method according to the invention, said bacteria, grown in phase 1 and 1' under low or moderate concentration of urea which did not promote the hydrolysis of urea, in phases 2, 2', 3 and 3' were homogeneously distributed and stabilized among the sand particles, then in step 4 they were induced to initiate the hydrolysis of urea.

The combination of the above steps resulted in the uniform distribution of the precipitated calcium carbonate in the subsurface layers.

Surprisingly, the method according to the invention overcomes the problems of the previous methods, in which the production of calcium carbonate begins when the bacteria come into contact with the surface layers, which in turn results in the impermeability of the material. surface layer and in preventing stabilization of subsurface layers. The method according to the invention ? then more efficient in controlling the urea hydrolysis process.

This method? useful for preventing the washing of the soil by floodwater and rain; prevent the evaporation of water due to calcification of the soil surface and promote the storage of water for a long time, thus changing the the biosystem towards the regrowth of plants, helping the production of nitrogenous substances and fertility? of the soil.

Additional advantages of the method lie in the use of non-pathogenic bacteria, non-contamination of the environment and humans, green science and lack of damage to the biosystem, non-dependence on petroleum products, chemicals and harmful substances.

Finally, the method ? quick to make and results in lasting work; ? low cost as it uses cheap and accessible raw materials.

For example, a rough cost comparison of raw material for microbial grouting and conventional chemical grouting suggested that the cost for microbial grouting is ? significantly more cheaper than chemical grouting: $0.5-9/m<3 >of soil vs $2-72/m<3 >of soil respectively.

Claims (10)

1. A method of increasing the concentration of calcium carbonate in a geomaterial comprising the following steps: 0) to isolate indigenous ureolytic bacteria from the geomaterial; 1) cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, in which the concentration of sodium acetate is ? of 100 mM, the concentration of urea ? from 0.01 M to 0.03 M, and the concentration of protein source from 18 g/l to 22 g/l; or 1') cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising urea and a protein source, in which the concentration of urea ? from 2 M to 3 M and the concentration of protein source? from 18 g/l to 22 g/l; 2) adding the suspension of the cultured indigenous ureolitic bacteria obtained in step 1) or 1') to a geomaterial; 3) add to the geomaterial treated in step 2) a solution comprising a calcium salt, a protein source, a bicarbonate salt, an ammonium salt and urea, and let the bacteria stabilize for a period of between 12 and 24 hours; or 2') premixing powders of a calcium salt, a protein source, a bicarbonate salt, an ammonium salt and urea with a geomaterial; 3') add to the premixed geomaterial in 2') the suspension of the cultured indigenous ureolitic bacteria obtained in phase 1) or 1') and let the bacteria stabilize for a period between 12 and 24 hours; 4) add to the geomaterial treated in 3) or 3') a solution comprising a calcium salt, a protein source, a bicarbonate salt, an ammonium salt and urea, continuously for at least 2 days. 2. The method according to claim 1, comprising 0) to isolate indigenous ureolytic bacteria from the geomaterial; 1) cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, in which the concentration of sodium acetate is ? of 100 mM, the concentration of urea ? from 0.01 M to 0.03 M, and the concentration of protein source from 18 g/l to 22 g/l; or 1') cultivating said indigenous ureolytic bacteria in non-sterile conditions on a medium comprising urea and a protein source, in which the concentration of urea ? from 2 M to 3 M and the concentration of protein source? from 18 g/l to 22 g/l; 2) adding the suspension of the cultured indigenous ureolitic bacteria obtained in step 1) or 1') to a geomaterial; 3) add to the geomaterial treated in 2) a solution comprising from 0.01M to 0.1M of a calcium salt, from 1.5 g/l to 2.5 g/l of a protein source, from 1g/l to 2, 1 g/l of a bicarbonate salt, 5 g/l to 10 g/l of an ammonium salt and 0.01M to 0.1M urea, and allow the bacteria to settle for 12 to 24 hours hours; or 2') premix with a powdered geomaterial 33.2-332 g of a calcium salt, 60-200 g of a protein source, 30-60 g of a bicarbonate salt, 150-500 g of an ammonium salt and 18- 180 g of urea per 1 square meter of geomaterial in depth? of 10 cm; 3') add to the pre-mixed geomaterial in step 2') the suspension of the cultured indigenous ureolitic bacteria obtained in step 1) or 1') and let the bacteria stabilize for a period of between 12 and 24 hours; 4) add to the geomaterial treated in phase 3) or 3') a solution comprising from 0.1M to 1M of a calcium salt, from 1.5 g/l to 2.5 g/l of a protein source, from 1g/ l to 2.1 g/l of bicarbonate salt, from 5 g/l to 10 g/l of ammonium salt and from 0.1M to 1M of urea, continuously for at least 2 days. The method according to claim 1 or 2, wherein the native urealytic bacteria are non-pathogenic bacteria constitutively expressing urease or in which urease is expressed only in the presence of urea; preferably selected from: Bacillus fortis, Bacillus lentus, Bacillus graminis, Sporosarcina pasteurii, Sporosarcina ureae, Bhargavaeace cembensis, Virgibacillus campisalis, Escherichia fergusonii, Proteus vulgaris, and their combination; more preferably selected from Bacillus fortis strain MK accession number KT862901, Bacillus lentus strain MK accession number KT862902, Bacillus lentus strain MK accession number KT862896, Sporosarcina pasteurii strain MK accession number KT862894, Bacillus graminis strain MK accession number KT862903, strain Bacillus graminis MK2 strain accession number KT862895, Bhargavaeace cembensis strain MK accession number KT862897, Virgibacillus campisalis strain MK accession number KT862898, Escherichia fergusonii strain MK accession number KT862899, Escherichia fergusonii strain MK2 accession number KT862900 and combinations thereof. 4. The method according to any one of the preceding claims, wherein the geomaterial is chosen from a structure with interconnected pores or fractures; preferably ? chosen from rock, such as conglomerate, breccia, sandstone, siltstone, shale, limestone, gypsum, dolostone, lignite; soil, such as gravel, sand, silt, clay with or without organic matter such as peat, sediment; fractured igneous or metamorphic rock, such as volcanic rock. 5. The method according to any one of the preceding claims, wherein to phase 1), 1'), the protein source? choice between meat extract, peptone, yeast extract; preferably the protein source? yeast extract, plus preferably yeast extract at a concentration of 20 g/l; and/or to phases 3) and 4) the protein source? choice between meat extract, peptone, yeast extract; preferably the protein source? yeast extract, plus preferably yeast extract at a concentration of 2 g/l; and/or to phase 2) and 3') the suspension of indigenous ureolytic bacteria contains from 10<7 >to 10<12 >CFU/mL; more preferably 10<9 >CFU/mL, and/or the suspension of indigenous ureolytic bacteria is added to the geomaterial in quantity? from 10 to 50 L/m2 with depth? of 10 cm, more preferably 30 L/m2 with depth? of 10 cm; and/or to phase 3) - the calcium salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or - the calcium salt ? calcium chloride, more preferably ? calcium chloride; and/or - the protein source solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or the protein source? yeast extract, plus preferably 2 g/l of yeast extract; and/or - the bicarbonate salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; and/or bicarbonate salt? preferably baking soda, pi? preferably baking soda; and/or - the ammonium salt solution is added to the geomaterial in an amount from 30-100 l/m2 of geomaterial with depth? of 10 cm, more preferably 70 l/m2 of geomaterial with depth? of 10 cm and/or the ammonium salt ? preferably ammonium chloride, pi? preferably ammonium chloride; and/or - the urea solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; and/or to phase 2') - powdered calcium salt is added to the geomaterial in an amount of 33.2-332 g/m² with depth? of 10 cm, preferably of 77.62 g/m2 with depth? of 10 cm; more preferably the calcium salt ? calcium chloride; and/or - the powdered protein source is added to the geomaterial in an amount of 60-200 g/m² with depth? of 10 cm, preferably of 140 g/m2 with depth? of 10 cm; more preferably the protein source? yeast extract; and/or - the bicarbonate salt powder is added to the geomaterial in a quantity of 30-60 g/m² with depth? of 10 cm, preferably 50 g/m² with depth? of 10 cm; more preferably bicarbonate salt? sodium bicarbonate; and/or - the powdered ammonium salt is added to the geomaterial in an amount of 150-500 g/m² with depth? of 10 cm, preferably of 350 g/m2 with depth? of 10 cm; more preferably the ammonium salt ? ammonium chloride; and/or - powdered urea is added to the geomaterial in an amount of 18-180 g/m² with depth? of 10 cm, preferably 42 g/m2 with depth? of 10 cm; and/or to phase 4) - the calcium salt solution is added to the geomaterial in an amount from 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; preferably the calcium salt ? calcium chloride, more preferably calcium chloride; and/or - the protein source solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm and/or the protein source? yeast extract, plus preferably 2 g/l of yeast extract; and/or - the bicarbonate salt solution is added to the geomaterial in an amount 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10 cm; and/or bicarbonate salt? preferably baking soda, pi? preferably baking soda; and/or - the ammonium salt solution is added to the geomaterial in an amount 30-100 l/m2 of geomaterial with depth? of 10 cm, more preferably 70 l/m2 of geomaterial with depth? of 10 cm and/or the ammonium salt ? preferably ammonium chloride, pi? preferably ammonium chloride; and/or - the urea solution is added to the geomaterial in quantity? equal to 30-100 l/m2 with depth? of 10 cm, more preferably 70 l/m2 with depth? of 10cm. 6. The method according to any one of the preceding claims wherein the suspension of the indigenous ureolitic bacteria added in step 2) and/or the solution of the ingredients added in steps 3 and 4, ?/are flowed or injected into the geomaterial under pressure; or sprayed, dripped, or dripped onto or into the geomaterial; or the geomaterial pu? be immersed in them; preferably sprayed onto or into the geomaterial, preferably sprayed into the sand; and/or the suspension of phase 3' indigenous ureolytic bacteria and/or the powders of phase 2' are mixed with the geomaterial. 7. The method according to any one of the preceding claims, which comprises the following steps 0) to isolate indigenous ureolytic bacteria from the geomaterial; 1) cultivating indigenous ureolytic bacteria, selected from Bacillus fortis strain MK accession number KT862901, Bacillus lentus strain MK accession number KT862902, Bacillus lentus strain MK accession number KT862896, Sporosarcina pasteurii strain MK accession number KT862894, Bacillus graminis strain MK accession number KT862903, Bacillus graminis strain MK2 accession number KT862895, Bhargavaeace cembensis strain MK accession number KT862897, Virgibacillus campisalis strain MK accession number KT862898, Escherichia fergusonii strain MK accession number KT862899, Escherichia fergusonii MK2 strain accession number KT862900 and a combination thereof, preferably consisting of Sporosarcina pasteurii MK strain accession number KT862894, under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium ? of 100 mM, the concentration of urea ? less than 0.03 M and more than 0.01 M, and the protein source ? yeast extract 20 g/l; 2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 meter square of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm; 3') mix the pre-mixed geomaterial in 2'), preferably sand, with a suspension of said indigenous cultured bacteria obtained in step 1) and let the bacteria stabilize for a period of between 12 and 24 hours; 4) add to the geomaterial treated in 3') a solution comprising from 0.1M to 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and 0. 1M to 1M urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days. 8. The method according to any one of the preceding claims, which comprises the following steps 0) to isolate indigenous ureolytic bacteria from the geomaterial; 1) culturing the indigenous ureolytic bacterium, consisting of the strain Sporosarcina pasteurii MK accession number KT862894 under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium acetate is ? of 100 mM and the concentration of urea ? less than 0.03M and more than 0.01M, and the concentration of yeast extract ? of 20 g/l; 2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 meter square of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm; 3') mix to the pre-mixed geomaterial in 2'), preferably sand, a suspension of said cultured indigenous bacteria obtained in phase 1) containing from 10<7> to 10<12>CFU/mL, plus? preferably 10<9 >CFU/mL, and allow the bacteria to settle for 12 to 24 hours; 4) add to the geomaterial treated in 3') a solution comprising from 0.1M to 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and 0. 1M to 1M urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days. 9. The method according to any one of the preceding claims, which comprises the following steps 0) to isolate indigenous ureolytic bacteria from the geomaterial; 1) culturing the indigenous ureolytic bacterium, consisting of the strain Sporosarcina pasteurii MK accession number KT862894 under non-sterile conditions on a medium comprising sodium acetate, urea and a protein source, where the concentration of sodium acetate is ? of 100 mM and the concentration of urea ? less than 0.03M and more than 0.01M, and the concentration of yeast extract ? of 20 g/l; 2') premix powders of 33.2 to 332 g of calcium chloride, 60-200 g of yeast extract, 150-500 g of NH4Cl, 30-60 g of NaHCO3 and 18 to 180 g of urea per 1 meter square of geomaterial in depth? of 10 cm, preferably for 1 square meter of sand in depth? of 10 cm; 3') mix to the pre-mixed geomaterial in 2'), preferably sand, a suspension of said cultured indigenous bacteria obtained in phase 1) containing from 10<7> to 10<12>CFU/mL, plus? preferably 10<9 >CFU/mL, and allow the bacteria to settle for 12 to 24 hours; 4) spraying on or in the geomaterial treated in 3') a solution comprising from 0.1M to 1M of calcium chloride, 2 g/l of yeast extract, 7g/l of NH4Cl, 1.25g/l of NaHCO3 and from 0.1M to 1M of urea, continuously for 3 days, preferably 4, or 5, or 6, or 7 days. 10. The method according to any one of the preceding claims, wherein in phase 3') the bacterial suspension is mixed with the geomaterial and the ingredients in phase 2'), preferably sand; and/or in phase 2') the ingredients are mixed with the geomaterial, preferably sand; and/or the bacteria are left to stabilize for 12-24 hours; and/or in phase 4) the ingredients are sprayed on the geomaterial, preferably sand from fixed nozzles for 3-7 days and at 175-185 ml/h.