Microorganism loaded aggregate and manufacturing methods

Abstract

The invention is directed to compositions, tools and methods for the manufacture of construction materials, masonry, solid structures and compositions to facilitate dust control. More particularly, the invention is directed to the manufacture of bricks, masonry and other solid structures using small amount of aggregate material that is pre-loaded with spores and/or vegetative bacterial cells.

Claims

1. A method of manufacturing a shelf-stable spore-loaded aggregate, the method comprising: (a) adding an aqueous medium to a collection of viable spore-forming bacteria to form an aqueous mixture; (b) incubating the aqueous mixture under conditions that promote the formation of spores and/or vegetative cells; (c) mixing the aqueous mixture incubated in (b) with aggregate particles; and (d) removing at least a portion of the aqueous medium to concentrate the spores or vegetative cells with the aggregate particles: (i) to a concentration of 10.sup.6 to 10.sup.15 cells/mL and (ii) until the mixture contains less than 50% liquid by weight, to provide the shelf stable spore-loaded aggregate, which remains at least 80% viable after months of storage.

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

3. The method of claim 1, wherein the aqueous medium does not contain urea.

4. The method of claim 1, wherein the viable spore-forming bacteria comprise one or more strains of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori, and/or a urease and/or a carbonic anhydrase producing microorganism.

5. The method of claim 1, wherein the incubating is performed at from 25-40° C.

6. The method of claim 1, wherein the incubating is performed from 6 hours to 6 days.

7. The method of claim 6, wherein the incubating is performed for 1-3 days.

8. The method of claim 1, wherein the conditions comprise a physiological pH.

9. The method of claim 1, wherein the mixing further includes addition of a binding agent.

10. The method of claim 9, wherein the binding agent comprises a polymer, a saccharide, a polysaccharide, a carbohydrate, a fatty acid, an oil, an amino acid, or a combination thereof.

11. The method of claim 9, wherein the binding agent promotes adhesion between spores and/or vegetative cells and the aggregate particles via hydrophobic bonds, hydrophilic bonds, ionic bonds, non-ionic bonds, covalent bonds, van der Waal forces, or a combination thereof.

12. The method of claim 1, wherein the aggregate particles comprise natural, non- natural, recycled or manufactured sand, ore, rock, stone, minerals, crushed materials, fractured glass, mine tailings, paper, waste materials, plastics, polymers, roughened materials, and/or any combinations thereof.

13. The method of claim 1, wherein the aggregate particles are in the form of beads, grains, strands, fibers, flakes, crystals, or combinations thereof.

14. The method of claim 1, wherein the aggregate particles comprise particles with a mesh size of 100 or smaller.

15. The method of claim 14, wherein the mesh size is 200 or smaller.

16. The method of claim 1, wherein the concentration of the spores produced in (b) is from about 10.sup.8 to about 10.sup.10 spores/mL.

17. The method of claim 1, wherein the aqueous medium stimulates spore formation.

18. The method of claim 1, wherein the aqueous medium maintains viability of the spore-forming bacteria without propagation.

19. The method of claim 1, wherein (d) concentrates the spores or vegetative cells with the aggregate until the mixture contains less than 10% liquid by weight.

Description

DESCRIPTION OF THE INVENTION

(1) The manufacture of masonry and other building materials using a process known as microbial induced calcite precipitation (MICP) has been extensively described in a number of U.S. Patent (e.g., see U.S. Pat. Nos. 8,728,365; 8,951,786; 9,199,880; and 9,428,418; each of which is incorporated in its entirety by reference). In these processes, urease-producing cells or urease enzymes are mixed with aggregate and incubated with urea and a calcium source. Calcite bonds form between aggregate particles resulting in a solid structure. Although the process allows for the manufacture of building materials, manufacturing generally requires standardization for the purpose of large-scale production.

(2) It has been surprisingly discovered that the manufacture of biologically-created solids can be standardized and, accordingly the manufacturing process enhanced. Standardization is achieved by adding an aqueous medium to a collection of viable spore-forming bacteria forming an aqueous mixture and incubating the aqueous mixture under conditions that promote spore formation. Spores of most urease-producing microorganisms are generally round, oval or slightly elongated with sizes from about 0.9 μm to 2.0 μm in length and from about 0.5 μm to about 1.0 μm in width. Spores and/or vegetative cells are then mixed with aggregate particles, preferably but not necessarily aggregate consistent with and/or similar to the bulk aggregate, forming a slurry and the slurry concentrated by the removal of at least a portion of the aqueous component, essentially the water, but not the spores and/or vegetative cells. Retention of spores and/or vegetative cells can be achieved by utilizing aggregate particles of a size or average size and composition that permits the transference of liquid such as water but retains spores and/or vegetative cells. These ultrafine aggregate particles can be maintained as a slurry or further liquid can be removed as desired to form a powder or solid structure.

(3) One embodiment of the invention is directed to a method for forming starter cultures of spores and/or vegetative cells and aggregate material for the manufacture of solid structures, construction materials, or the manufacture of compositions to utilize for dust control applications (e.g., U.S. Pat. No. 8,951,786 issued 10 Feb. 2015; U.S. Pat. No. 9,428,418 issued 30 Aug. 2016; U.S. Patent Application Publication No. 2016/0264463 entitled “Compositions and Methods for Dust Control and the Manufacture of Construction Materials” published 16 Sep. 2016; U.S. Patent Application Publication No. 2016/0362334; each of which is entirely incorporated by reference). Spores and vegetative cells can be cultured from spore-producing bacterial and/or may be previously prepared and, preferably, under conditions that specifically induce the formation or spores or vegetative cells. The resulting solution or spore-containing or cell-containing culture is mixed with aggregate particles forming a slurry. Spores and/or vegetative cells (microorganisms) are concentrated from the slurry by removing at least a portion of liquid. Preferably the aggregate particles are largely or completely of a mesh size of 100 or smaller (particles of 150 μm or smaller), more preferably 200 or smaller (particles of about 75 μm or smaller) and more preferably 300 or smaller (particles of about 38 μm or smaller). Generally, particle sizes are measured or determined as average sizes. Water and dissolved aqueous materials can be removed and the microorganisms remain within the aggregate. In this way aggregate material can be loaded with a desired amount or quantity of microorganisms and stored. The microorganism-loaded aggregate particles can be maintained as a slurry, or dried as a powder or solid form. As the mixture contains aggregate and microorganisms, the mixture is relative resistant to variations in temperature or most any other external conditions, and therefore can be maintained for long periods of time. In this way, large numbers of spores and/or vegetative cells can be maintained to coordinate large manufacturing operations.

(4) In a first step, spore-forming bacteria are cultured, preferably under conditions that promote spore and/or vegetative cell formation. Culture conditions include an aqueous medium comprising one or more of salts, amino acids, proteins, peptides, carbohydrates, saccharides, polysaccharides, fatty acids, oil, vitamins and minerals. Preferably, the aqueous medium does not contain urea, which will stimulate spore formation of the bacteria. Preferred spore-forming bacteria comprise one or more strains of Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis, Bacillus megaterium, Helicobacter pylori, and/or any urease and/or carbonic anhydrase producing microorganism. Bacteria are cultured in the aqueous medium, preferably at incubation is at a physiological pH and at temperatures of from about 25-40° C. Preferably incubation is performed from about 6 hours to about 6 days, more preferably for about 1-3 days, or as short a time as necessary to generate the desired number of spores and/or vegetative cells per bacterium.

(5) Preferably spore formation or vegetative cell formation is induced, although an induction step is not required, and the microorganisms may be centrifuged or otherwise concentrated, and preferably resuspended into a paste with media or another suitable liquid that maintains the microorganisms without inducing further growth and/or proliferation (a status solution). Alternatively, microorganisms may be need mixed with aggregate without concentration, which may be preferable for manufacturing batches of vegetative cells.

(6) Following spore-formation or vegetative cell formation as desired, cultures are mixed with aggregate particles. Aggregate particles may comprise natural, non-natural, recycled or manufactured sand, ore, crushed rock or stone, minerals, crushed or fractured glass, mine tailings, paper, waste materials, waste from a manufacturing process, plastics, polymers, roughened materials, and/or combinations thereof, and may be in the form of beads, grains, strands, fibers, flakes, crystals, or combinations thereof. Preferably the aggregate particles comprise particles with a mesh size of 100 or smaller (particles of about 150 μm or smaller), more preferably with a mesh size is 200 or smaller (particles of about 75 μm or smaller), or more preferably with a mesh size of 300 or smaller (particles of about 38 μm or smaller).

(7) Preferably the aqueous mixture of spores and/or vegetative cells and/or the aggregate is combined with a binding agent that promotes the adhesion or retention of microorganisms and aggregate. Adhesion may be between microorganisms and aggregate via hydrophobic bonds, hydrophilic bonds, ionic bonds, non-ionic bonds, covalent bonds, van der Waal forces, or a combination thereof. Binding agents include, but are not limited to one or more of polymers, saccharides, polysaccharides, carbohydrates, fatty acids, oils, amino acids, or combinations thereof. Preferred binding agents are nontoxic and/or biodegradable and also preferably harmless to the spores and do not interfere or otherwise hinder eventual germination of spores or proliferation of vegetative cells.

(8) Preferably the aqueous component and mixture is removed is by evaporation and/or filtration, such as, for example, heat-assisted evaporation, pressure-assisted filtration, and/or vacuum-assisted filtration. Following evaporation and/or filtration, the slurry or aggregate particles and microorganisms contains from about 10.sup.6 to about 10.sup.14 spores and/or cells/ml, preferably from about 10.sup.8 to about 10.sup.12, and more preferably from about 10.sup.9 to about 10.sup.11. The aqueous component can be further removed or removed entirely without hard to the spores and/or vegetative cells and the dried powder or block stored for future use in starting a culture of urease-producing bacteria.

(9) Spore-containing aggregate material has a very long shelf life with greater than 80% viability after months and/or years with proper storage conditions. Vegetative-containing aggregate has a somewhat shorter shelf life with greater than 80% remaining viable after months and longer with proper storage conditions.

(10) Another embodiment of the invention is directed to a composition comprising spore-loaded aggregate made by the methods of the invention. Preferably aggregate particles are of a mesh size of 100 or smaller (particles of about 150 μm or smaller), 200 or smaller (particles of about 75 μm or smaller), or 300 or smaller (particles of about 38 μm or smaller). Also preferably, the composition contains a binding or retention agent. The binding agent promotes adhesion between spores and/or vegetative cells and aggregate particles and/or the retention agent increases the size of aggregate particles and/or spores and/or vegetative cells, which promotes their retention.

(11) Preferably the composition contains less than about 50% liquid by weight, more preferably less than about 10% liquid by weight, and more preferably less than about 5% liquid by weight. Preferred compositions contain from about 10.sup.19 to about 10.sup.15 spores and/or vegetative cells/ml.

(12) Another embodiment of the invention is directed to methods of manufacturing construction material comprising combining in any order compositions of the invention with urea, calcium, an aggregate material, and an incubation medium forming a mixture. The mixture is incubated under conditions that promote formation of calcium carbonate which forms the solid structure of aggregate materials. Preferred solid structures include, for example, construction materials such as bricks, thin bricks, pavers, panels, tile, veneer, cinder, breeze, besser, clinker or aerated blocks, counter- or table-tops, design structures, blocks, or a solid masonry structure. Preferably the calcium is provided from calcium chloride, calcium acetate, calcium phosphate, calcium carbonate, calcium lactate, calcium nitrate, or a calcium salt. Also preferably the aggregate material comprises natural, non-natural, recycled or manufactured sand, ore, crushed rock or stone, minerals, crushed or fractured glass, wood, ash, foam, basalt, fibers, mine tailings, paper, waste materials, waste from a manufacturing process, plastics, polymers, roughened materials, and/or combinations thereof. Solid structures can be formed in a formwork or extruded as desired. Extruded aggregate retains a basic shape upon extrusion that solidifies over time into a solid structure at a desired hardness.

(13) The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

EXAMPLES

Example 1

Spore Production

(14) Sporosarcina pasteurii spores were produced from vegetative cells in culture media.

Example 2

Spore Loading

(15) Spores (approximately 1 μm in diameter) were directly loaded into aggregate fines either by (1) vacuum-assisted or pressure-assisted filtration (or simply gravity assisted), or (2) evaporation. Alternatively, spore cultures may be concentrated and stored refrigerated in phosphate buffered saline (PBS) until ready for loading.

(16) Method 1: Vacuum-Assisted Filtration

(17) 1. Whole spore cultures (2 L) were mixed with 1 kg ultrafine (<75 μm avg. diameter) manufactured aggregates.

(18) 2. A filtering apparatus was applied and the vacuum or a pressure engaged to facilitate liquid clearance.

(19) 3. Loaded fines were harvested and dried completely.

(20) Method 2: Evaporation

(21) 1. Whole spore cultures (2 L) were mixed with 1 kg ultrafine (<75 μm avg. diameter) mining aggregates.

(22) 2. Spore aggregate mixtures were spread evenly over maximum surface area and dried under circulating air.

(23) 3. Loaded fines were harvested and dried completely. Spore loading aggregate from Method 1 demonstrated high retention of dormant spores within crude aggregate. Method 2 yielded a similar spore to aggregate distribution.

(24) Other embodiments and uses of the invention will be apparent to those skilled in the art from 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 specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.