Biological sintering without heat or pressure

Abstract

To provide a novel tool, composition, and method for the manufacture of building materials.SOLUTION: The invention is directed to compositions, tools and methods of biological sintering involving the enzymatic break-down and reformation of calcium carbonate. In particular, the invention is directed to the manufacture of bricks, masonry and other solid structures, dust control, and the construction of roads, paths, and other solid surfaces using one or more enzymes that precipitate and / or dissolve calcium carbonate.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 806,346, filed on February 15, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention is directed to compositions, tools, and methods for biological sintering involving the enzymatic breakdown and re - formation of calcium carbonate. In particular, the present invention relates to the use of one or more enzymes that precipitate and / or dissolve calcium carbonate in the manufacture, dust - proofing of bricks, masonry, and other solid structures, and the construction of roads, paths, and other solid surfaces.

Background Art

[0003] Conventional brick and concrete structures rely heavily on the combustion of natural resources such as coal and wood. This reliance consumes large amounts of energy resources and, similarly, releases large amounts of carbon dioxide, thus depending greatly on limited energy sources. Alternatives to these conventional processes include a process known as microbially induced calcite precipitation (MICP). MICP involves mixing urease and urea as an energy source with an aggregate such as sand, for example. This enzyme catalyzes the production of ammonia and carbon dioxide, raising the pH level of the composition. The second enzyme, carbonic anhydrase, facilitates the transition of carbon dioxide to carbonate anions. The increase in pH forms a mineral "precipitate" that combines calcium cations and carbonate anions. The particles present in the mixture function as nucleation sites, attracting mineral ions from calcium to form calcite crystals. Mineral growth fills the gaps between the biocementation or sand particles that bind to each other. Preferably, the particles include gaps that are at least 5 microns wide, although the width can be larger or smaller as needed. The resulting material exhibits a composition and physical properties similar to those of naturally formed stone, brick, or other solid structures. The hardness can be determined in advance based on at least the structure of the initial components and the desired pore size.

[0004] Examples of enzyme-producing bacteria capable of dissolving calcium carbonate include Alphaproteobacteria, Betaprobacteria, Gammaprobactreia, Firmicutes, or Actinobacteria. Examples of enzyme-producing bacteria capable of biocementation include Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, or Helicobacter pylori, but appropriate precautions must be taken with pathogenic strains. Combinations of any of these strains, as well as functional mutants, mutations, and genetically engineered strains can likewise be used. The bacterial composition includes a nutrient medium for maintaining and / or enabling good cell growth and proliferation. Various types of nutrient media for cells, particularly for the bacterial cells of the present invention, are known and commercially available, and include at least minimal media (or transport media) commonly used for transport to maintain viability without growth, and yeast extract and molasses, which are commonly used for growth and proliferation.

[0005] This method of manufacturing construction materials by induced cementation exhibits low embodied energy and can occur at atmospheric pressure and over a wide range of temperatures. Depending on the ambient temperature and conditions and the available aggregate content, it may be determined whether pure enzymes, lyophilized enzymes, or live cells are utilized as starting components. Generally, live cells are used at relatively warm temperatures where mild weather conditions exist, while pure enzymes may be advantageous under relatively extreme conditions of cold or heat. The introduction of bioengineered building units using sand aggregate and naturally induced cementation offers a natural alternative that can be manufactured locally and is potentially environmentally friendly. Since little or no heating is required, significant energy savings can be achieved in both cost and efficiency.

[0006] Another advantage of MICP is that its process can be utilized on both small and large scales and can be easily automated. The bulk content of the stone manufacturing process of the present invention can be almost all materials available locally, including rock, sand, gravel, and almost all types of stone. Stone processing such as crushing or breaking into pieces can also be carried out locally. Therefore, transportation costs and expenses can be minimized. The composition of the present invention (which can be provided lyophilized and hydrated on-site), the brick frame (if not available in another way), and, if necessary, the instructions for use are all that is required to be provided. If transportation is required, this represents only a fraction of the delivery cost, especially compared to the current costs associated with the delivery of conventional concrete.

[0007] Another advantage of the MICP process is that it mainly utilizes minerals, MICP, and loose aggregates (such as sand) to manufacture "grown" construction materials such as bricks. It is not only possible to manufacture bricks and other construction materials, but also to fix the bricks themselves in place with cement at the intended location, "cement-fix" the bricks to each other and / or to other materials, thereby forming buildings, support structures or members, walls, roads, and other structures.

[0008] Biologically grown bricks and stones do not require the conventional use of Portland cement mortar, thereby reducing carbon dioxide in the atmosphere by providing an alternative to conventionally manufactured construction materials with high embodied energy. By using cells to naturally induce mineral precipitation and combining it with local aggregates and rapid manufacturing methods, it becomes possible to produce local, ecological, and economical building materials used throughout the world's construction industry.

[0009] MICP can be utilized to manufacture almost all forms of bricks, blocks, or solid structures used in construction, but an efficient method for large-scale manufacturing has not yet been developed. Therefore, there is a need for a rapid and convenient process that provides consistency in the production of economical and environmentally safe stones. Furthermore, the initial components required for MICP are not always readily available. The calcium source is often only available in the form of solid calcium carbonate. Therefore, it is necessary to obtain calcium.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new tools, compositions, and methods for the manufacture of building materials.

Means for Solving the Problems

[0011] One embodiment of the present invention is directed to a method comprising providing a first aqueous medium comprising a microorganism that expresses an enzyme that dissolves calcium carbonate, combining the first aqueous medium with calcium carbonate under conditions that promote the activity of the enzyme that dissolves calcium carbonate, and collecting calcium ions and / or free carbon.

[0012] In a preferred embodiment, the aqueous medium contains one or more of salts, amino acids, proteins, peptides, carbohydrates, sugars, polysaccharides, fatty acids, oils, vitamins, and minerals for the growth and proliferation of microorganisms, or is maintained in a minimal medium until use. Preferably, the microorganism includes one or more species, subspecies, strains, or serotypes of Alphaproteobacteria, Betaprobacteria, Gammaprobactreia, Firmicutes, or Actinobacteria. Preferably, the microorganism includes one or more species, subspecies, strains, or serotypes of Variovorax, Klebsiella, Pseudomonas, Bacillus, Exiguobacterium, Microbacterium, Curtobacterium, Rathayibacter, CellFimi2, Streptomyces, and / or Raoultella.

[0013] Another embodiment of the present invention is directed to a method of forming calcium carbonate. The method includes providing a second aqueous medium containing a microorganism that expresses an enzyme for forming calcium carbonate, combining the second aqueous medium with collected calcium ions and / or collected free carbon under conditions that promote the activity of the enzyme for forming calcium carbonate, and forming calcium carbonate. The calcium ions and / or free carbon are collected by providing a first aqueous medium containing a microorganism that expresses an enzyme for dissolving calcium carbonate, combining the first aqueous medium with calcium carbonate under conditions that promote the activity of the enzyme for dissolving calcium carbonate, and collecting the calcium ions and / or free carbon.

[0014] Preferably, the microorganism includes one or more species, subspecies, strains, or serotypes among Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori, and / or microorganisms that produce urease and / or carbonic anhydrase. In a preferred embodiment, combining includes adding a binder. Preferably, the binder includes a polymer, saccharide, polysaccharide, carbohydrate, protein, peptide, fatty acid, oil, amino acid, or a combination thereof.

[0015] Another embodiment of the present invention is directed to a composition comprising a microorganism that expresses an enzyme that dissolves calcium carbonate and an aggregate material.

[0016] Another embodiment of the present invention is directed to a method for manufacturing a construction material. The method includes providing a first aqueous medium comprising a microorganism that expresses an enzyme that dissolves calcium carbonate, combining the first aqueous medium with calcium carbonate under conditions that promote the activity of the enzyme that dissolves calcium carbonate to form calcium ions and / or free carbon, combining the calcium ions and / or free carbon with a second aqueous medium comprising a microorganism that expresses an enzyme that forms calcium carbonate, and forming calcium carbonate.

[0017] Another embodiment of the present invention is directed to a method for manufacturing construction materials. The method includes providing an aqueous medium comprising a consortium of microorganisms expressing an enzyme that dissolves calcium carbonate and a microorganism expressing an enzyme that forms calcium carbonate; combining the medium with calcium carbonate to form calcium ions and / or free carbon, and forming calcium carbonate.

[0018] Another embodiment of the present invention is directed to a composition comprising a microorganism expressing an enzyme that dissolves and forms calcium carbonate and an aggregate material. Preferably, the calcium carbonate constitutes a construction material. In a preferred embodiment, the construction material constitutes bricks, thin bricks, paving materials, panels, tiles, veneers, charcoal shells, breezes, besser, clinkers or aeration blocks, counter tops or table tops, designed structures, blocks, solid stone structures, bridge piers, foundations, beams, walls or slabs (e.g., concrete).

[0019] Another embodiment of the present invention is directed to a composition comprising a mixture of microorganisms, where one group of microorganisms dissolves calcium carbonate upon exposure to a first condition, and another group of microorganisms forms calcium carbonate under a second condition (where the second condition can be the same as, substantially the same as, or different from the first condition). Preferably, the composition further comprises an aggregate material (e.g., limestone, sand, silicate material, or a combination thereof), and preferably, the aggregate material is from about 10 weight percent to about 95 weight percent of the composition (e.g., about 20 weight percent, about 30 percent, about 40 percent, about 50 percent, about 60 percent, about 70 percent, about 80 percent, about 90 percent). A relatively high percentage of aggregate is common for use, although a relatively low percentage of aggregate can be a concentrated form of the composition for storage or transportation. Preferably, the first microorganism as cells and / or spores comprises one or more species, subspecies, strains, or serotypes of Alphaproteobacteria, Betaprobacteria, Gammaprobactreia, Firmicutes, or Actinobacteria, and furthermore, preferably, the first microorganism constitutes from about 10 weight percent to about 40 weight percent of the composition. Preferably, the second microorganism as cells and / or spores comprises one or more species, subspecies, strains, or serotypes of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, or Helicobacter pylori.Preferably, the combined first and second microorganisms constitute from about 10 weight percent to about 100 weight percent (e.g., about 15 percent, about 20 percent, about 25 percent, about 30 percent, about 35 percent, about 40 percent, about 45 percent, about 50 percent, about 55 percent, about 60 percent, about 65 percent) of the composition. A relatively high percentage of the non-aggregate components of the composition is common for storage or transportation purposes, while a relatively low percentage of non-aggregate components is more common for use. Preferably, the composition may not contain an aggregate material, where the aggregate material is added prior to use as needed for a particular application. Preferably, the composition contains water at about 25 weight percent or less, 20 weight percent or less, 10 weight percent or less, about 5 weight percent or less, or about 2 weight percent or less. The composition can also further contain components that support the germination and / or growth of the first and / or second microorganisms, such as nutrients, carbohydrates, polysaccharides, buffers, salts, stabilizers, preservatives, and the like. Preferably, the first and second microorganisms continue to survive in the composition for 3 months or more, 6 months or more, 9 months or more, 12 months or more, 24 months or more, or 36 months or more.

[0020] Other embodiments and advantages of the present invention are described in part in the following description and may in part be apparent from this description or learned from the practice of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The production of stone and other building materials using a process known as microbially induced calcite precipitation (MICP) is widely described in many U.S. patents (see, e.g., U.S. Patent Nos. 8,728,365, 8,951,786, 9,199,880, and 9,428,418; each of which is incorporated by reference in its entirety). In these processes, urease-producing cells or urease enzyme are mixed with an aggregate and incubated with a urea and calcium source. Calcite bonding forms between the aggregate particles and results in a solid structure. While this process enables the production of building materials, production generally requires standardization for large-scale manufacturing purposes.

[0022] Surprisingly, it has been found that calcium can be collected from the dissolution of calcium carbonate by microorganisms that produce enzymes that dissolve calcium carbonate and / or the enzymes themselves, thereby forming calcium ions and carbon ions. Examples of microorganisms that produce enzymes that dissolve calcium carbonate include the following: species, subspecies, strains or serotypes of Alphaproteobacteria, Betaprobacteria, Gammaprobactreia, Firmicutes or Actinobacteria, such as Variovorax, Klebsiella, Pseudomonas, Bacillus, Exiguobacterium, Microbacterium, Curtobacterium, Rathayibacter, CellFimi2, Streptomyces and / or Raoultella species, subspecies, strains or serotypes. The calcium ions and potentially free carbon ions produced by these enzymes can be utilized by microorganisms that express enzymes that produce calcium carbonate.Examples of microorganisms that produce enzymes for producing calcium carbonate include the following: Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori and / or species, subspecies, strains or serotypes of microorganisms that produce urease and / or carbonic anhydrase.

[0023] The process of biological sintering without using heat and pressure utilizes microorganisms that produce enzymes that decompose calcium carbonate as a calcium source that can be used to reform calcium carbonate using microorganisms that produce enzymes that form calcium carbonate. In a similar manner, the dissolution of calcium also releases carbon that can be used as a carbon source for calcium carbonate formation.

[0024] Calcium and calcium carbonate produced by enzymes can be standardized, and accordingly the manufacturing process is enhanced. Standardization is achieved by adding an aqueous medium to a collection of live bacteria to form an aqueous mixture and incubating the aqueous mixture under conditions that promote growth. In the case of cells that dissolve calcium carbonate, the cells are mixed with calcium carbonate solids. When forming calcium carbonate, the cells or enzymes are mixed with raw materials for forming calcium carbonate. It is possible to mix the nutrient cells or enzymes with particles (e.g., calcium carbonate particles or aggregate particles that match and / or are similar to the solid structures to be formed) to form a slurry, and the slurry can be concentrated by removing at least a portion of the aqueous component (essentially water and not cells). Retention of the cells can be achieved by utilizing aggregate particles of a size or average size and composition that allow the movement of a liquid such as water while retaining the cells. These ultrafine aggregate particles can be maintained as a slurry or, if desired, further liquid can be removed to form a powder or solid structure.

[0025] One embodiment of the present invention is directed to a method for forming a starter culture of microorganisms that dissolve calcium carbonate and / or microorganisms that form calcium carbonate. Water and dissolved aqueous materials can be added or removed, and the microorganisms can be added or removed as needed. The microorganisms can be maintained as a slurry or dried into a powder form or solid form. Preferably, the microorganisms are maintained in an aqueous or dried form that is relatively resistant to temperature changes or most other external conditions and can thus be maintained for a long period of time. In this way, a large number of microorganisms can be maintained to adjust large-scale manufacturing operations.

[0026] In a first step, the spore-forming bacterium is cultured under conditions that preferably promote spore and / or vegetative cell formation. The culture conditions include an aqueous medium containing one or more of salts, amino acids, proteins, peptides, carbohydrates, sugars, polysaccharides, fatty acids, oils, vitamins, and minerals. Preferred microorganisms that dissolve calcium carbonate include Variovorax, Klebsiella, Pseudomonas, Bacillus, Exiguobacterium, Microbacterium, Curtobacterium, Rathayibacter, CellFimi2, Streptomyces, and / or Raoultella. Preferred microorganisms that form calcium carbonate include one or more strains of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori, and / or microorganisms that produce urease and / or carbonic anhydrase. The microorganism is maintained in minimal medium until use and then cultured in an aqueous medium, preferably at physiological pH and a temperature of about 25-40°C during incubation. Preferably, the incubation is carried out for about 6 hours to about 6 days, more preferably about 1-3 days, or for as short a time as necessary to produce the desired number of spores and / or vegetative cells per bacterium.

[0027] Preferably, sporulation or vegetative cell formation is induced, but the induction stage is not necessary, and the microorganism can be subjected to centrifugation or concentrated by other methods, and preferably, it can be resuspended in a paste having a medium or another suitable liquid for maintaining the microorganism without inducing further growth and / or proliferation (status solution). Alternatively, the microorganism may need to be mixed with the aggregate without concentration, which may be preferred for producing a batch of vegetative cells. Preferably, the composition further comprises an aggregate material (e.g., limestone, sand, silicate material, or a combination thereof). Preferably, the aggregate can be included in the composition in an amount of about 10 weight percent to about 99 weight percent (e.g., about 20 weight percent, about 30 percent, about 40 percent, about 50 percent, about 60 percent, about 70 percent, about 80 percent, about 90 percent, about 95 percent). A relatively high percentage of the aggregate is common for use, but a relatively low percentage of the aggregate can be a concentrated form of the composition for storage or transportation. Preferably, the combined first and second microorganisms comprise about 10 weight percent to about 70 weight percent or more of the composition (e.g., about 15 percent, about 20 percent, about 25 percent, about 30 percent, about 35 percent, about 40 percent, about 45 percent, about 50 percent, about 55 percent, about 60 percent, about 65 percent). A relatively high percentage of the non-aggregate components of the composition is common for storage or transportation applications, but a relatively low percentage of the non-aggregate components is more common for use. Preferably, the composition may not contain an aggregate material, where the aggregate material is added prior to use as needed for a particular application. Typically, the first and second microorganisms are present in relatively equal amounts. However, in applications where there is a large amount of calcium carbonate to be decomposed, the first microorganism can be dominant, and conversely, in cases where there is a large amount of calcium carbonate to be formed, the second microorganism can be dominant. The respective amounts can be determined by those skilled in the art as needed for a particular application.Preferably, the composition contains about 25 weight percent or less water, 20 weight percent or less water, 10 weight percent or less water, about 5 weight percent or less water, or about 2 weight percent or less water. The composition can also further contain components that support the germination and / or growth of the first and / or second microorganisms, such as nutrients, carbohydrates, polysaccharides, buffers, salts, stabilizers, preservatives, and the like.

[0028] Optionally, following sporulation or vegetative cell formation, the culture is mixed with the aggregate particles. The aggregate particles can include natural sand, non-natural sand, recycled sand or crushed sand, ore, crushed rock or stone, minerals, crushed or broken glass, mining debris, paper, waste materials, waste rock from manufacturing processes, plastics, polymers, roughened materials, and / or combinations thereof, and can be in the form of beads, grains, strands, fibers, flakes, crystals, or combinations thereof. Preferably, the aggregate particles contain particles with a mesh size of 100 or less (particles of about 150 μm or less), more preferably particles with a mesh size of 200 or less (particles of about 75 μm or less), or even more preferably particles with a mesh size of 300 or less (particles of about 38 μm or less).

[0029] Preferably, an aqueous mixture of spores and / or vegetative cells and / or aggregates is combined with a binder that promotes adhesion or retention of the microorganism and the aggregate. Adhesion can occur between the microorganism and the aggregate via hydrophobic bonding, hydrophilic bonding, ionic bonding, non-ionic bonding, covalent bonding, van der Waals forces, or combinations thereof. Examples of binders include, but are not limited to, one or more of polymers, saccharides, polysaccharides, carbohydrates, peptides, proteins, fatty acids, oils, amino acids, or combinations thereof. Preferred binders are non-toxic and / or biodegradable, and furthermore, are preferably harmless to the spores and do not inhibit or otherwise interfere with the eventual germination of the spores or the growth of the vegetative cells. Furthermore, preferably, the composition does not contain poisons, toxic substances, or components that pose a risk to the viability of the microorganism or to individuals handling the composition or the final product.

[0030] Preferably, the aqueous components and mixtures are removed by evaporation and / or filtration (e.g., evaporation assisted by heat, filtration assisted by pressure, and / or filtration assisted by reduced pressure). After subjecting to evaporation and / or filtration, the slurry or aggregate particles and microorganisms contain from about 10 6 to about 10 14 spores and / or cells, preferably from about 10 8 to about 10 12 spores and / or cells, more preferably from about 10 9 to about 10 11 spores and / or cells per mL. The aqueous component can be further removed or completely removed without causing difficulties to the spores and / or vegetative cells and without causing difficulties to the dried powder or block that is stored for future use when initiating the culture of urease-producing bacteria.

[0031] The aggregate containing spores has a long storage life. Preferably, the storage life results in a survival rate of more than about 80% (preferably, a survival rate of about 90%, about 95% or about 99%) after storage for about 3 months, about 6 months, about 9 months or about 12 months, or results in a survival rate of more than about 80% (preferably, a survival rate of about 90%, about 95% or about 99%) after storage for about 1 year, about 2 years, about 3 years, about 4 years or about 5 years. The aggregate containing vegetative cells has a somewhat shorter storage life and has a survival rate of more than about 80% (preferably, a survival rate of about 90%, about 95% or about 99%) after storage for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months or about 6 months.

[0032] Another embodiment of the present invention is directed to a composition comprising a spore-added aggregate produced by the method of the present invention. Preferably, the aggregate particles are particles with a mesh size of 100 or less (particles of about 150 μm or less), particles with a mesh size of 200 or less (particles of about 75 μm or less) or particles with a mesh size of 300 or less (particles of about 38 μm or less). Furthermore, preferably, the composition contains a binder or a holding agent. The binder promotes adhesion between the spores and / or vegetative cells and the aggregate particles, and / or the holding agent increases the size of the aggregate particles and / or the spores and / or vegetative cells and promotes their retention.

[0033] Preferably, the composition contains less than about 50% by weight of liquid, more preferably less than about 10% by weight of liquid, and even more preferably less than about 5% by weight of liquid. A preferred composition contains about 10 10 ~ about 10 15 spores and / or vegetative cells per mL.

[0034] Another embodiment of the present invention is directed to a method for manufacturing construction materials, where the method includes combining a calcium carbonate solution with microorganisms and / or enzymes, and subsequently utilizing calcium and / or carbon obtained from the dissolution in the production of calcium carbonate using the microorganisms and / or enzymes. Solid calcium carbonate can be formed in a mold or extruded as needed. The extruded calcium carbonate retains its basic shape when extruded and solidifies over time into a solid structure with the desired hardness.

[0035] The following examples illustrate embodiments of the present invention and should not be regarded as limiting the scope of the present invention.

Examples

[0036] Example 1 Microbial production for dissolving calcium carbonate Cultures of Variovorax, Klebsiella, Pseudomonas, Bacillus, Exiguobacterium, Microbacterium, Curtobacterium, Rathayibacter, CellFimi2, Streptomyces, and Raoultella were generated from natural sources and established cultures obtained from the American Type Culture Collection (ATCC). The cultures were maintained in minimal medium (e.g., a pH-balanced salt solution for maintaining viability without promoting growth or germination) until ready for use.

[0037] Example 2 Dissolution of calcium carbonate The microorganism of Example 1 is mixed with calcium carbonate in solid form to form a slurry, to which are added components required for growth and proliferation for a specific culture (for example, this may include carbohydrates, sugars, polysaccharides, carbohydrates, fatty acids, lipids, vitamins, proteins, peptides, amino acids, salts, pH buffers, minerals and / or additional components). The microorganism dissolves the calcium carbonate, forming calcium ions and free carbon.

[0038] Example 3 Microbial production for dissolving calcium carbonate Cultures of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori were generated from natural sources and established cultures obtained from the "American Type Culture Collection (ATCC)". The cultures are maintained in minimal medium (for example, a pH-balanced salt solution to maintain viability without promoting growth or germination) until ready for use.

[0039] Example 4 Formation of calcium carbonate The microorganism of Example 3 is mixed with the calcium ions and free carbon produced according to Example 2, to which are added components required for growth and proliferation for a specific culture (for example, this may include carbohydrates, sugars, polysaccharides, carbohydrates, fatty acids, lipids, vitamins, proteins, peptides, amino acids, minerals, salts, pH buffers and / or additional components). The microorganism forms calcium carbonate.

[0040] Other embodiments and uses of the invention will be apparent to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are hereby specifically and completely incorporated by reference. The term "comprising," as used heretofore, is intended to include the terms "consisting of" and "consisting essentially of." Further, the terms "comprising," "including," and "containing" are not intended to be limiting. The specification and examples are to be considered exemplary only, and it is intended that the true scope and spirit of the invention be indicated by the following claims.

Claims

1. providing a first aqueous medium comprising a microorganism expressing an enzyme that dissolves calcium carbonate; combining said first aqueous medium with calcium carbonate under conditions that promote the activity of an enzyme that dissolves calcium carbonate; and collecting calcium ions and / or free carbon; A method comprising:

2. 10. The method of claim 1, wherein the aqueous medium comprises one or more of salts, amino acids, proteins, peptides, carbohydrates, sugars, polysaccharides, fatty acids, oils, vitamins and minerals.

3. 2. The method of claim 1, wherein the microorganism comprises one or more species, subspecies, strains or serotypes of Alphaproteobacteria, Betaprobacteria, Gammaprobacteria, Firmicutes or Actinobacteria.

4. 2. The method of claim 1, wherein the microorganism comprises one or more species, subspecies, strains or serotypes of Variovorax, Klebsiella, Pseudomonas, Bacillus, Exiguobacterium, Microbacterium, Curtobacterium, Rathayibacter, CellFimi2, Streptomyces and / or Raoultella.

5. 1. A method for forming calcium carbonate comprising the steps of: providing a second aqueous medium containing a microorganism expressing an enzyme that forms calcium carbonate; combining said second aqueous medium with said collected calcium ions and / or free carbon and nitrogen sources collected according to the method of claim 1 under conditions promoting activity of an enzyme that forms calcium carbonate; and forming calcium carbonate; The method comprising:

6. The microorganism is selected from the group consisting of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori, and the like.

6. The method of claim 5, comprising one or more species, subspecies, strains or serotypes of H. pylori and / or urease and / or carbonic anhydrase producing microorganisms.

7. The method of claim 5 , wherein the combining comprises adding a binder.

8. The method of claim 7 , wherein the binder comprises a polymer, a sugar, a polysaccharide, a carbohydrate, a fatty acid, an oil, an amino acid, or a combination thereof.

9. 6. The method of claim 5, wherein combining the first aqueous medium is carried out substantially with combining the second aqueous medium.

10. 1. A method of producing a material, comprising: providing a first aqueous medium comprising a microorganism expressing an enzyme that dissolves calcium carbonate; combining said first aqueous medium with calcium carbonate under conditions promoting activity of an enzyme that dissolves calcium carbonate to form calcium ions and / or free carbon; combining the calcium ions and / or free carbon with a second aqueous medium containing a microorganism expressing an enzyme that forms calcium carbonate; and forming calcium carbonate; The method comprising:

11. 11. The method of claim 10, wherein the calcium carbonate comprises a construction material.

12. 12. The method of claim 11, wherein the construction material comprises a brick, a thin brick, a paving material, a panel, a tile, a veneer, a charcoal, a breeze, a besser, a clinker or aerated block, a countertop or a tabletop, an engineered structure, a block, a solid masonry structure, a pier, a foundation, a beam, a wall or a slab.

13. 11. The method of claim 10, wherein the first aqueous medium and / or the second aqueous medium comprises one or more of salts, amino acids, proteins, peptides, carbohydrates, sugars, polysaccharides, fatty acids, oils, vitamins and minerals.

14. 1. A method for producing a construction material, comprising: Providing an aqueous medium comprising a microorganism expressing an enzyme that dissolves calcium carbonate and a microorganism expressing an enzyme that forms calcium carbonate; and combining said aqueous medium with calcium carbonate under conditions promoting the activity of an enzyme that dissolves calcium carbonate to produce calcium ions and / or free carbon; wherein the microorganism expressing an enzyme that forms calcium carbonate utilizes the calcium ions and / or free carbon to form calcium carbonate.

15. 15. The method of claim 14, wherein the calcium carbonate constitutes a construction material.

16. 16. The method of claim 15, wherein the construction material comprises a brick, a thin brick, a paving material, a panel, a tile, a veneer, a charcoal, a breeze, a besser, a clinker or aerated block, a countertop or tabletop, an engineered structure, a block, a solid masonry structure, a pier, a foundation, a beam, a wall or a slab.

17. A composition comprising a first microorganism expressing an enzyme that dissolves calcium carbonate and a second microorganism expressing an enzyme that forms calcium carbonate.

18. 18. The composition of claim 17, wherein the first microorganism comprises one or more species, subspecies, strains or serotypes of Alphaproteobacteria, Betaprobacteria, Gammaprobacteria, Firmicutes or Actinobacteria.

19. 18. The composition of claim 17, wherein the second microorganism comprises one or more species, subspecies, strains or serotypes of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori.

20. 20. The composition of claim 17, wherein the first microorganism and / or the second microorganism comprises a spore.

21. The composition of claim 17 further comprising aggregate.

22. 22. The composition of claim 21, wherein the aggregate comprises sand, crushed sand, fine stone, crushed concrete, crushed brick, limestone, silicate material, or combinations thereof.

23. 20. The composition of claim 17, wherein the first microorganism comprises from about 1.0 percent to about 50 percent by weight of the composition suspended in a medium that maintains viability and does not promote growth or proliferation of the microorganism.

24. 20. The composition of claim 17, wherein the second microorganism comprises from about 1.0 percent to about 40 percent by weight of the composition suspended in a medium that maintains viability and does not promote the growth or proliferation of the microorganism.

25. 22. The composition of claim 21, wherein the aggregate comprises from about 10 percent to about 95 percent by weight of the composition.

26. 20. The composition of claim 17 comprising less than about 10 percent by weight water.

27. 20. The composition of claim 17 comprising less than about 5 percent by weight water.

28. 20. The composition of claim 17 comprising less than about 2 percent by weight water.

29. 20. The composition of claim 17, comprising an ingredient that promotes germination and / or growth of the first microorganism and / or the second microorganism.

30. 30. The composition of claim 29, wherein the ingredients include nutrients, carbohydrates, polysaccharides, stabilizers, preservatives, buffers and / or salts.

31. 20. The composition of claim 17, wherein the first and second microorganisms remain viable for about six months or more.

32. 20. The composition of claim 17, wherein the first and second microorganisms remain viable for about 12 months or more.

33. 20. The composition of claim 17, wherein the first and second microorganisms remain viable for about 24 months or more.

34. 20. The composition of claim 17, wherein the first microorganism and / or the second microorganism comprises a spore.

35. 20. The composition of claim 17, further comprising calcium carbonate.