Improved Concrete Composition and Method for Producing the Same

Abstract

The present invention relates to compositions for improving concrete using biochemically produced microorganisms and / or by-products synthesized by the microorganisms. The present invention also relates to methods for improving the performance of concrete using the microbial strains and / or by-products thereof.

Description

Technical Field

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 979,161, filed Feb. 20, 2020, the contents of which are incorporated by reference herein.

Background Art

[0002] Concrete is one of the more common building materials than steel, plastic, and wood. Current methods of manufacturing concrete are based on mixing cement, sand, and water with additional chemicals that modify the properties of the concrete. An example of an additive is a chemical surfactant that acts as a plasticizer. The use of this plasticizer is particularly beneficial when manufacturing high-strength concrete and fiber-reinforced concrete.

[0003] As the moisture concentration in the concrete mixture increases, the concrete strength decreases. For every 1% increase in moisture concentration, the final strength of the concrete decreases by 5%. However, without adequate water in the concrete mixture, the concrete cannot be flowed or handled into the desired form.

[0004] Concrete plasticizers are added to concrete to reduce the amount of water required for handling. Plasticizers are typically added at a concentration of 0.1% - 0.4%. At these concentrations of plasticizer, the moisture required to maintain the plasticity of the concrete is reduced by 5% - 15%.

[0005] The cultivation of microorganisms such as bacteria, yeast, or fungi is important for the production of various useful bioproducts. Microorganisms play important roles in, for example, the food industry, pharmaceutical industry, agriculture, mining, environmental remediation, and waste management. There is great potential for the expanded use of microorganisms in a wide range of industries.

[0006] Interest in microbial biosurfactants is steadily increasing due to their diversity, environmentally friendly properties, selectivity, performance under extreme conditions, and potential uses in environmental protection. Biosurfactants are microbial-derived chemicals that reduce the interfacial tension between phases. Sophorolipids (SLP) are biosurfactants belonging to the class of glycolipids produced by non-pathogenic yeasts. SLP can be used in agriculture, food preservation, biomedicine, cosmetics, and other industries.

Summary of the Invention

Means for Solving the Problems

[0007] The present invention provides unique and advantageous uses of microorganisms and growth by-products of microorganisms such as biosurfactants. In certain embodiments, the present invention provides microbial-based products and their use in improved concrete compositions.

[0008] Specifically, in a preferred embodiment, the present invention provides a cost-effective and environmentally friendly approach to improving various properties of concrete. Among the advantages is that these methods can be implemented over a wide range of environmental conditions, including underwater and regions with large temperature variations.

[0009] In certain embodiments, the present invention provides compositions that improve various properties of concrete by the application of microorganisms and / or their growth by-products. The microorganisms and / or their growth by-products can be added to water, cement, aggregates, and other necessary elements of the concrete product during the initial mixing of the concrete. Alternatively, the microorganisms and / or growth by-products can be applied at any time after the initial pouring of the concrete, including when the concrete has fully solidified.

[0010] In certain embodiments, the present invention utilizes yeast strains and / or their growth by-products. In one embodiment, the microorganisms used in the compositions and methods of the present invention are biosurfactant-producing yeasts. The present invention, for example, culturedStarmerella bombicola ATCC 22214 Provide microbial-based products and / or growth products of such microorganisms. Further, the present invention relates to cultured Wickerhamomyces anomalus and / or provide microbial-based products containing growth by-products thereof. The microorganisms are in various growth stages including vegetative or spore forms.

[0011] In certain embodiments, the present invention further comprises one or more additional components such as, for example, accelerators, binders, corrosion inhibitors, air entraining agents, crystalline admixtures, pigments, plasticizers, superplasticizers, pumping aids, retarders, water reducers, shrinkage reducing agents, hydration control admixtures, alkali-silica reactivity inhibitors, moisture-proof admixtures, magnetic permeability reducing admixtures, gas-forming admixtures, non-segregating admixtures, foaming admixtures, and / or workability admixtures.

[0012] In certain embodiments, the present invention provides a method for improving the performance of concrete by adding a composition according to the present invention comprising a microbial culture and / or microbial growth by-products to a concrete mixture and / or applying it to the surface of hardened concrete. The performance of the concrete can be improved, for example, by reducing the amount of water used in the concrete composition, increasing the plasticity of the concrete, reducing the porosity of the concrete, changing the setting time of the concrete, and / or increasing the resistance to biological and / or abiotic stresses.

Embodiments for Carrying Out the Invention

[0013] The present invention provides the advantageous use of microorganisms and growth by-products of microorganisms such as biosurfactants. In certain embodiments, the present invention provides microbial-based products and their use in improved concrete compositions.

[0014] In certain embodiments, the methods and compositions described herein utilize microorganisms and / or microbial growth by-products as admixtures for concrete. In one embodiment, the microorganisms used in the method of the present invention are biosurfactant-producing yeasts.

[0015] In certain embodiments, the present invention utilizes yeast strains and / or their growth by-products. The present invention provides, for example, a microbial-based product comprising cultured Wickerhamomyces anomalus yeast and / or its growth by-products. Further, the present invention provides a microbial-based product comprising cultured Starmerella Kluyveromyces, preferably Starmerella ( Candida ) bombicola and / or its growth by-products.

[0016] In a preferred embodiment, the present invention provides a method for improving one or more properties of concrete by applying one or more microorganisms and / or microorganism-derived biosurfactants to the concrete. In certain embodiments, the method comprises applying one or more biosurfactants derived from a yeast strain, such as Starmerella bombicola or Wickerhamomyces anomalus to the concrete.

[0017] In certain embodiments, the method may also comprise applying the microorganism itself and / or its by-products together with one or more admixtures conventionally used in concrete.

[0018] In certain embodiments, the methods and compositions described herein utilize microbial growth by-products as an applied product during or after concrete hardening. The microorganisms and / or by-products penetrate the concrete or remain on the concrete surface.

[0019] In one embodiment, the compositions according to the present invention are obtained by small-scale to large-scale culture processes. These culture processes include, but are not limited to, submerged culture / fermentation, surface culture, solid-state fermentation (SSF), and combinations thereof.

[0020] Advantages include that the present invention can be used without releasing large amounts of inorganic compounds into the environment. Further, the present compositions and methods are biodegradable and utilize toxicologically safe components. Accordingly, the present invention can be used as a "green" treatment in all possible coatings (and other applications).

[0021] Selected Definitions As used herein, "microbe-based composition" means a composition containing components produced as a result of the growth of microbes or other cell cultures. Accordingly, a microbe-based composition includes the microbes themselves and / or microbe growth by-products. The cells may be in a vegetative state or spore form, or a mixture of both. The cells may be planktonic, in a biofilm form, or a mixture of both. Growth by-products are, for example, metabolites, cell membrane components, expressed proteins, and / or other cell components. The cells may be intact or lysed. In some embodiments, the cells are present in the microbe-based composition together with the broth in which they grew. The cells are present, for example, at a concentration of at least 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , or 1×10 11 or more cells per milliliter of the composition.

[0022] The present invention further provides a "microbe-based product", which is a product actually applied to achieve a desired result. A microbe-based product is simply a microbe-based composition collected from a microbial culture process. Alternatively, a microbe-based product may contain additional added components. These additional components include, for example, appropriate carriers such as buffers, water, added nutrients to support further microbial growth, and / or agents that facilitate the tracking of the composition in the microbe and / or the environment to which it is applied. Also, a microbe-based product may contain a mixture of microbe-based compositions. Further, a microbe-based product may contain one or more components of a microbe-based composition that have been treated by any method, including but not limited to filtration, centrifugation, dissolution, drying, purification, etc.

[0023] As used herein, an "isolated" or "purified" biosurfactant or other biochemically active substance of microbial origin substantially does not contain other compounds such as cell materials naturally associated therewith. As used herein, "isolated" means that the strain is removed from the environment in which it naturally occurs. Thus, an isolated strain may exist, for example, as a biologically pure culture or as spores (or other forms of the strain).

[0024] "Metabolite" refers to any substance produced by metabolism (e.g., growth by-products) or a substance necessary to participate in a specific metabolic process. Examples of metabolites include, but are not limited to, biosurfactants, biopolymers, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, trace elements, and amino acids.

[0025] "Regulate" means to change (increase or decrease). Such changes are detected by standard known methods as described herein.

[0026] The ranges provided herein are understood to be abbreviated for all values within those ranges. For example, a range of 1 to 50 includes any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all decimal values between the above integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” extending from either endpoint of the range are particularly contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 can include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0027] “Decrease” means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0028] “Increase” means a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0029] “Reference” means a standard or control condition.

[0030] As used herein, a “biofilm” is a complex aggregate of microorganisms such as bacteria, in which cells adhere to each other and / or to a surface using an extracellular polysaccharide matrix. The cells in a biofilm are physiologically distinct from planktonic cells of the same organisms, which can be single cells that float or swim in a liquid medium.

[0031] As used herein, "surfactant" refers to a compound that reduces the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants act, for example, as detergents, wetting agents, emulsifiers, foaming agents, and / or dispersing agents. Surfactants produced by microorganisms are called "biosurfactants".

[0032] As used herein, "biological stress" is the pressure on a composition and / or object that results from a living organism such as bacteria, fungi, or barnacles. "Biological stressors" differ from "abiotic stressors" in that "abiotic stressors" result from abiotic factors such as UV radiation, freeze-thaw cycles, or salt water.

[0033] As used herein, "accelerator", "cement accelerator", "accelerating admixture", and "concrete accelerator" are components used in concrete, cement, mortar, rendering, or screed that accelerate the setting time of a concrete composition. Accelerators can be used to counteract the effects of adverse weather conditions that can slow down or damage the setting time of concrete, especially during cold periods, regardless of the presence of precipitation. Examples of accelerators include calcium nitrate, calcium nitrite, calcium formate, calcium chloride, triethanolamine, and sodium thiocyanate.

[0034] As used herein, "binder", "adhesive", "resin", and "epoxy" are components used in concrete that can bond concrete to existing structures such as concrete, plaster, and stucco. Without a binder, fresh concrete cannot be adhered to a hardened concrete structure. Examples of binders include polyvinyl chloride, polyvinyl acetate, acrylic, and butadiene-styrene copolymer.

[0035] As used herein, "corrosion inhibitor" is a component used in concrete that can prevent premature failure of reinforced concrete by restricting the reaction of chlorides and atmospheric CO2 with cement. Chlorides from sources such as deicing salts can reach the embedded steel and cause rust. Examples of corrosion inhibitors include amino alcohols, calcium nitrite, and sodium monofluorophosphate.

[0036] As used herein, "air-entraining agent" is a compound used in concrete to create minute air bubbles in the composition. The air-entraining agent allows air to expand and contract during temperature cycles, improves the workability and cohesion of the concrete, and promotes the development of air pockets in the concrete to enhance durability by reducing segregation and bleeding of the concrete. Examples of air-entraining agents include salts of wood resins, synthetic detergents, salts of sulfonated lignins, salts of petroleum acids, salts of proteinaceous substances, fatty acids and resin acids and their salts, alkylbenzene sulfonates, and salts of sulfonated hydrocarbons.

[0037] As used herein, "crystalline admixture" or "permeability reducer" reduces the permeability of the concrete to which it is added. In certain examples, the concrete becomes almost impermeable to water by adding an appropriate amount of crystalline admixture. Examples of crystalline admixtures include latex and calcium stearate.

[0038] As used herein, "plasticizer", "superplasticizer", "workability admixture", and "water-reducing admixture" are added to concrete to reduce the amount of water required to handle it, increase fluidity, and / or reduce the water-to-cement ratio. Some examples of chemical plasticizers include lignosulfates, polyglucose esters, carbohydrates, and hydroxylated carboxylic acids. There are also superplasticizers containing modified lignosulfates and sulfonated naphthalene formaldehyde, which are superior to plasticizers in their ability to enable the workability of concrete with up to a 30% reduction in water in water.

[0039] As used herein, a "pumping aid" is a lubricating substance that enables concrete to flow easily through pipes, channels, drums, or other objects for a concrete transport chamber or a holding chamber. Examples of pumping aids include organic and synthetic polymers, organic flocculants, organic emulsions of paraffin, coal tar asphalt, acrylics, bentonite, calcined silica, and hydrated lime.

[0040] As used herein, a "retarder" is a substance that slows the setting time of concrete. Some examples of retarders include lignin, borax, sugar, and tartaric acid and salts. Similarly, a "hydration control admixture" also slows the setting time of concrete, but the hydration control mixture is often more forgiving after the evaporation process has begun. A hydration control admixture can be compared to a time-release cap cell.

[0041] According to the present invention, a "shrinkage reducing device" when concrete solidifies restricts the shrinkage of concrete. Some examples of shrinkage reducing agents include polyoxyalkylene alkyl ethers and propylene glycol.

[0042] As used herein, an "alkali-silica reactivity inhibitor" inhibits swelling that can occur over the life of concrete by inhibiting the reaction between an alkali cement paste and silica. Sodium silicate results from the reaction in producing a hygroscopic gel that increases in volume when exposed to water. Some examples of alkali silica reaction inhibitors include barium salts, lithium nitrate, lithium carbonate, and lithium hydroxide.

[0043] As used herein, a "moisture-proof" admixture prevents or hinders the penetration of moisture into concrete. Moisture is commonly used to prevent moisture from the surrounding environment from penetrating into the concrete. These moisture sources are the soil or sand that surrounds underground walls, concrete slabs, or the concrete of sidewalks. Moisture-proofing is often the formation of an external coating on the concrete, as opposed to an additive that is mixed throughout the concrete composition. Some of the materials that can be used to make concrete proof against moisture include calcium or ammonium stearate, calcium or ammonium oleate, butyl stearate, and various petroleum products.

[0044] As used herein, a "waterproof" admixture is similar to moisture-proofing, but is designed to prevent penetration by liquid water, particularly in environments with a particularly high water table, frequent flooding, or within a pool. Waterproofing materials are also applied to the outer surface of the concrete. Some of the materials that can be used for waterproof concrete include rubberized asphalt coatings, bentonite, rubber-based coatings, and urethane coatings.

[0045] As used herein, a "gas-forming" admixture is a substance that expands the concrete before it hardens. The most common gas-forming admixtures are aluminum powder, activated carbon, and hydrogen peroxide. These added chemicals form bubbles of hydrogen gas within the concrete. In addition to resisting the settlement of the concrete, the presence of hydrogen gas also resists the bleeding of the concrete. Concrete bleeding occurs when water is pushed upward and the denser cement particles sink. Gas-forming admixtures can also be used to make lightweight concrete.

[0046] As used herein, a "non-segregating" admixture is often added to concrete for use in water to mitigate the effects of water pressure and friction caused by the movement of water. The non-segregating admixture improves the cohesion of the concrete and prevents the cement paste from being washed away. Concrete is self-compacting. Cellulose and acrylic polymers can be used in non-segregating compositions.

[0047] As used herein, a "foaming" admixture makes the concrete lighter and less dense by adding other chemicals such as surfactants or hydrolyzed proteins to the concrete composition. Concrete having a foaming admixture is often used for void filling as opposed to a structural support.

[0048] As used herein, "aggregate" or "construction aggregate" is one of the three basic components for making concrete together with water and cement. Aggregate is a particulate material. Aggregate usually constitutes the majority of the concrete composition and includes sand, stone, gravel, glass, silica, blast furnace slag, recycled concrete, rock, or other related components.

[0049] The transitional phrase "comprising", which is synonymous with "having" or "containing", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not "substantially affect the basic and novel characteristics of the invention" recited in the claim. The use of the term "comprising" is intended to embrace embodiments "consisting" or "consisting essentially" of the recited elements.

[0050] Unless otherwise defined or clear from the context, the term "or" as used herein is understood to be inclusive. Unless otherwise defined or clear from the context, when used herein, the terms "a" and "the" are understood to be singular or plural.

[0051] Unless otherwise defined or clear from the context, the term "about" as used herein is understood to be within the normal tolerance range in the art, for example, within two standard deviations of the average. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0052] The listing of a list of chemical groups in any definition of a variable described herein includes the definition of that variable as any single group or combination of the listed groups. The listing of embodiments of a variable or aspect herein includes that embodiment as any single embodiment, in combination with any other embodiment or part thereof.

[0053] Yeast strains according to the present invention In preferred embodiments, the present invention utilizes biochemically producing yeasts. The yeast is a natural or genetically modified microorganism. For example, the yeast is transformed with a specific gene to exhibit specific characteristics. Also, the yeast may be a mutant of a desired strain. As used herein, "mutant" means a strain, genetic variant, or subtype of a reference microorganism, and the mutant has one or more genetic mutations (e.g., point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation, or repeat expansion) compared to the reference microorganism. Procedures for generating mutants are well known in the field of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.

[0054] Yeast and fungal species suitable for use according to the present invention include, but are not limited to, Acaulospora , Aspergillus, Aureobasidium (e.g., A. pullulans ), Blakeslea , Candida (e.g., C. albicans , C. apicola ), Cryptococcus , Debaryomyces (e.g., D. hansenii ), Entomophthora , Fusarium , Hanseniaspora (e.g., H. uvarum ) 、Hansenula , Issatchenkia , Kluyveromyces , Mortierella , Mucor (e.g., M. piriformis) , Meyerozyma (e.g., M. guilliermondii ), Penicillium , Phycomyces , Phycomyces , Pichia (e.g., P. anomala , P. guilliermondii , P. occidentalis , P. kudriavzevii ), Pseudozyma (e.g., P. aphidis ), Rhizopus , Saccharomyces ([[]] S. cerevisiae , S. boulardii sequela , S. torula ), Starmerella (e.g., S. bombicola ), Torulopsis , Thraustochytrium , Trichoderma (e.g., T. reesei , T. harzianum , T. virens ), Ustilago (e.g., U. maydis ), Wickerhamomyces (e.g., W. anomalus ), Williopsis and Zygosaccharomyces (e.g., Z. bailii ) are exemplified.

[0055] In some embodiments, the yeast is a "killer yeast", which means a strain of yeast characterized by the secretion of a toxic protein or glycoprotein that is immune to the strain itself. The exotoxin secreted by killer yeast can kill other strains of yeast, fungi, or bacteria, for example. Killer yeasts include, but are not limited to, Wickerhamomyces spp.(W. anomalus ) Pichia 、 Hansenula 、 Saccharomyces 、 Hanseniaspora 、( Hanseniaspora uvarum ) Ustilago maydis 、 Debaryomyces hansenii 、 Candida spp( C. albicans 、 C. rugosa 、 C. tropicalis 、 C. lipolytica 、 C. torulopsis ) Starmerella spp.( S. bombicola ) Cryptococcus 、 Kluyveromyces 、 Torulopsis 、 Ustilago 、 Williopsis 、 Zygosaccharomyces etc. can be mentioned.

[0056] In a specific embodiment, the microorganism is Starmerella spp. yeast and / or Candida spp. yeast, for example, Starmerella ( Candida ) bombicola 、 Candida apicola 、 Candida batistae 、 Candida floricola 、 Candida riodocensis 、 Candida stellate and / or Candida kuoi and is. In a specific embodiment, the microorganism is Starmerella bombicola 、for example, strain ATCC22214 and is.

[0057] In a specific preferred embodiment, the microorganism is Wickerhamomyces anomalus and is.

[0058] For example, other microbial strains containing other fungal strains that can accumulate a considerable amount of, for example, glycolipid biosurfactants or other useful metabolites such as carbohydrates, polyols, lipids, esters, and / or proteins can be used according to the present invention. Other useful metabolites and / or microbial components according to the present invention include mannan protein, beta-glucan, and others having bioemulsifying properties and surface / interface tension reducing properties.

[0059] Growth of yeast according to the present invention The present invention utilizes methods for culturing microorganisms and methods for producing microbial metabolites and / or other by-products of microbial growth. Microbial culture systems typically use submerged culture fermentation, but surface culture and hybrid systems can also be used.

[0060] As used herein, "fermentation" refers to the growth of cells under controlled conditions. The growth can be aerobic or anaerobic. Unless the context requires otherwise, this term is intended to encompass both the growth phase and the product biosynthesis phase of the process.

[0061] As used herein, "broth", "culture broth", or "fermentation broth" refers to a culture medium containing at least nutrients. In the case of broth after the fermentation process, the broth also contains microbial growth by-products and / or microbial cells.

[0062] In one embodiment, the present invention provides materials and methods for producing biomass (e.g., viable cell mass), extracellular metabolites (e.g., low molecular weight and secreted proteins), residual nutrients, and / or intracellular components (e.g., enzymes and other proteins).

[0063] The microbial growth vessel used in accordance with the present invention is any fermenter or culture reactor for industrial use. As used herein, the terms "reactor", "bioreactor", or "fermentation reactor" include a fermentation apparatus consisting of one or more vessels and / or towers or piping arrangements. Examples of such reactors include, but are not limited to, continuous stirred tank reactors (CSTRs), immobilized cell reactors (ICRs), trickle bed reactors (TBRs), bubble columns, gas lift fermenters, static mixers, or other vessels or other devices suitable for gas-liquid contact. In some embodiments, the bioreactor may include a first growth reactor and a second fermentation reactor. Thus, when adding a substrate to a bioreactor or fermentation reaction, appropriate addition to one or both of these reactors is included.

[0064] In one embodiment, the container may have a function control / sensor or be connected to a function control / sensor to measure important factors in the culture process such as pH, oxygen, pressure, temperature, stirrer shaft output, humidity, viscosity, and / or microbial density and / or metabolite concentration.

[0065] In a further embodiment, the container can also monitor the growth of microorganisms in the container (e.g., measurement of cell number and growth phase). Alternatively, daily samples may be taken from the container and subjected to counting by techniques known in the art such as dilution plating techniques. Dilution plating is a simple technique used to estimate the number of cells in a sample. This technique can also provide an indicator for comparing different environments or treatments.

[0066] In one embodiment, the method includes supplementing the culture with a nitrogen source. The nitrogen source is, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and / or ammonium chloride. These nitrogen sources may be used alone or in combination of two or more.

[0067] This culture method can oxygenate the growing culture. According to one embodiment, slow movement of air is utilized to remove low-oxygen-containing air and introduce oxygenated air. The oxygenated air may be ambient air that is replenished daily by a mechanism including an impeller for mechanical stirring of the liquid and an air sparger for supplying bubbles to the liquid for dissolution of oxygen in the liquid.

[0068] This method further includes supplementing the culture with a carbon source. The carbon source is typically a carbohydrate such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and / or maltose, an organic acid such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and / or pyruvic acid, an alcohol such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and / or glycerol, or an oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil, and / or linseed oil. These carbon sources may be used alone or in combination of two or more.

[0069] In one embodiment, growth factors and micronutrients of the microorganism are included in the medium. In one embodiment, inorganic salts may also be included. In certain embodiments, the culturing method may further include adding additional acid and / or antibacterial agent to the liquid medium before and / or during the culturing process. The antibacterial agent or antibiotic is used to protect the culture from contamination. Further, an antifoaming agent may be added to prevent the formation and / or accumulation of foam when gas is generated during culturing.

[0070] The pH of the mixture shall be suitable for the target microorganism. A buffer solution and pH regulators such as carbonates and phosphates can be used to stabilize the pH near the preferred value. When metal ions are present at high concentrations, the use of a chelating agent in the liquid medium is necessary.

[0071] The microorganism can be grown in a planktonic form or as a biofilm. In the case of a biofilm, the container can have a substrate on which the microorganism can grow in a biofilm state. Also, this system may have the ability to apply, for example, stimuli (such as shear stress) that promote and / or improve biofilm growth characteristics.

[0072] In one embodiment, the method for culturing microorganisms is carried out at about 5°C to about 100°C, preferably 15°C to 60°C, more preferably 25°C to 50°C. In a further embodiment, the culturing may be carried out continuously at a constant temperature. In other embodiments, the culturing may be exposed to varying temperatures.

[0073] In one embodiment, the method and the apparatus used in the culturing process are sterile. Culturing devices such as reactors / containers may be separated from, or connected to, a sterilization unit, such as an autoclave. Also, the culturing device may have a sterilization unit that sterilizes in situ before inoculation is initiated. Air can be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the container. In other embodiments, the medium may be pasteurized or, optionally, no heat may be applied at all, and it may be utilized to control bacterial growth without the use of low water activity and low pH.

[0074] The biomass content of the fermentation broth is, for example, 5 g / l to 180 g / l or more. In one embodiment, the solids content of the broth is 10 g / l to 150 g / l.

[0075] In one embodiment, the present invention further provides a method for producing microbial metabolites such as ethanol, polyols, esters, lactic acid, beta-glucan, proteins, peptides, metabolic intermediates, polyunsaturated fatty acids, biosurfactants, and lipids. The metabolite content produced by this method is, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0076] Microbial growth by-products produced by the target microorganism may be retained in the microorganism or secreted into the liquid medium. In other embodiments, the method for producing microbial growth by-products may further include the step of concentrating and purifying the target microbial growth by-products. In further embodiments, the liquid medium may contain a compound that stabilizes the activity of the microbial growth by-products.

[0077] In a preferred embodiment, the microbial growth by-product is a biosurfactant. Specific biosurfactants according to the present invention include, for example, low molecular weight glycolipids (GL), lipopeptides (LP), flavolipids (FL), phospholipids, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes.

[0078] In one embodiment, the microbial biosurfactant is a glycolipid such as rhamnolipid (RLP), sophorolipid (SLP), trehalose lipid or mannosylerythritol lipid (MEL). In one embodiment, the microbial biosurfactant is a lipopeptide such as iturin, fengycin or surfactin.

[0079] In one embodiment, the yeast-based composition comprises a blend of any of these biosurfactants. Preferably, the blend comprises sophorolipid and optionally one or more of mannosylerythritol lipid, surfactin, iturin and / or rhamnolipid.

[0080] In certain embodiments, the microbial growth by-products are carbohydrates, polyols, lipids, glycolipids, esters, and / or proteins. These components are by-products as fermentation waste. The growth by-products can also be residual chemicals resulting from yeast cell death.

[0081] In certain embodiments, the purified compound is at least 60% by weight of the compound of interest. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the compound of interest. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, such as column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis.

[0082] Methods and apparatuses for culturing microorganisms and producing microbial by-products can be carried out in batch, semi - continuous, or continuous processes.

[0083] In one embodiment, all of the microbial culture composition is removed at the end of the culture (e.g., when a desired cell density or the density of a particular metabolite in the broth is achieved). In this batch process, an entirely new batch is started at the time of collection of the first batch.

[0084] In other embodiments, only a portion of the fermentation product is removed at any given time. In this embodiment, the biomass with live cells remains in the vessel as an inoculum for a new culture batch. The composition removed may be cell - free broth or may contain cells. In this way, a semi - continuous apparatus is created.

[0085] Advantages include that this method does not require complex equipment or high energy consumption. The microorganism of interest can be cultured on - site, either on a small or large scale, and can be utilized even when mixed with the medium. Similarly, microbial metabolites can also be produced in large quantities at the required site.

[0086] Advantages include that microorganism - based products can be manufactured remotely. Microbial growth facilities can be operated without electricity, for example, by utilizing sunlight, wind power, and / or hydropower.

[0087] Production of Yeast - based Products One yeast - based product of the present invention is simply a fermentation broth containing yeast and / or microbial metabolites produced by yeast and / or any residual nutrients. The fermentation product can be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard extraction and / or purification methods or techniques described in the literature.

[0088] The yeast in the yeast - based product is in an active form, an inactive form, or a mixture thereof. The yeast - based product is used without further stabilization, preservation, and storage. Among the advantages, the direct use of these yeast - based products preserves the high viability of the microorganisms, reduces the potential for contamination from foreign substances and unwanted microorganisms, and maintains the activity of microbial growth by - products.

[0089] In one embodiment, a first yeast fermentation product designated "Star3+" can be obtained through the culture of yeast, for example, using a modified form of solid - state fermentation. Wickerhamomyces anomalus through the culture of Wickerhamomyces anomalus is often related to the production of food and grains and is effective in producing various solvents, enzymes, toxins, triglycerides, and biosurfactants (e.g., phospholipids). The culture can be grown on substrates having a sufficient surface area for yeast attachment and propagation, such as rice, soybeans, chickpeas, pasta, oatmeal, or beans. The entire fermentation medium containing growing yeast cells and their growth by - products (e.g., enzymes, solvents, and / or biosurfactants) can be recovered, for example, after culturing at 25 - 30 °C for 3 - 5 days. The culture is blended with the substrate, milled, and / or micronized and optionally dried. This includes the Star3+ product. For example, a composition containing 1×10 10 ~10 12 cells / gram can be diluted, for example, up to 10, 50, 100, 500, or 1,000 times before mixing with other components.

[0090] In one embodiment, the yeast fermentation product can be obtained through the immersion culture of Wickerhamomyces anomalus . The fermentation broth after culturing at 25-30 °C for 7 days contains a yeast cell suspension and, for example, at least 1-100 g / L, 2-80 g / L, 3-60 g / L, 4-40 g / L, 5-20 g / L or 6-10 g / L of biosurfactant.

[0091] In one embodiment, the yeast fermentation product can also be obtained through the culture of biosurfactant-producing yeast, Starmerella bombicola . This strain is effective for the production of glycolipid biosurfactants such as SLP. The fermentation broth after culturing at 25 °C for 5 days contains a yeast cell suspension and, for example, at least 1-150 g / L, 2-120 g / L, 3-100 g / L, 4-80 g / L, 5-60 g / L or 6-50 g / L of glycolipid biosurfactant.

[0092] In certain embodiments, the biosurfactant of the composition comprises one or more glycolipid biosurfactants. In certain preferred embodiments, the glycolipid is sophorolipid.

[0093] Sophorolipids are, for example, Starmerella the glycolipid biosurfactants produced by yeast of the genus Cryptococcus, in some embodiments, Wickerhamomyces anomalus . SLP consists of the disaccharide sophorose linked to a long-chain hydroxy fatty acid. They can contain a partially acetylated 2-O-β-D-glucopyranosyl-D-glucopyranose unit β-glycosidically linked to 17-L-hydroxyoctadecanoic acid or 17-L-hydroxy-Δ9-octadecenoic acid. The hydroxy fatty acid generally has 16 or 18 carbon atoms and contains one or more unsaturated bonds. Further, the sophorose residue is acetylated at the 6- and / or 6'-positions. The fatty acid carboxyl group can be free (acidic or chain form (general formula 1)) or internally esterified at the 4"-position (lactone form (general formula 2)). S. bombicola is S. bombicolaIt produces a specific enzyme called lactone esterase, which catalyzes the esterification of chain SLP to produce lactone SLP.

[0094] [Chemical formula] In the formula, R 1 and R 1′ are independently a saturated hydrocarbon chain, or a single or multiple, especially a single unsaturated hydrocarbon chain, having 8 to 20 carbon atoms, especially 12 to 18 carbon atoms, more preferably 14 to 18 carbon atoms, and can be linear or branched, and represent those that can contain one or more hydroxy groups. R 2 and R 2′ include and are independently a hydrogen atom, or a saturated alkyl functional group, or a single or multiple, especially a single unsaturated alkyl functional group, having 1 to 9 carbon atoms, more preferably 1 to 4 carbon atoms, and can be linear or branched, and represent those that can contain one or more hydroxy groups. R 3 R 3′ R 4 and R 4′ independently represent a hydrogen atom or an acetyl group.

[0095] Sophorolipids may be in a purified form or a mixture of fermentation products. Sophorolipids can be added to the concrete composition at a concentration of 0.001 to 90% by weight (wt%), preferably 0.01 to 50 wt%, more preferably 0.1 to 20 wt%. In other embodiments, the purified SLP may be combined with an acceptable carrier in that the SLP is present at a concentration of 0.001 to 50% (v / v), preferably 0.01 to 20% (v / v), more preferably 0.02 to 5% (v / v).

[0096] The yeast and / or broth obtained from yeast growth can be removed from the growth vessel and transferred, for example, via a pipe for immediate use.

[0097] The yeast fermentation product can include yeast cells and fermentation broth, or can include fermentation broth separated from yeast cells. In one embodiment, biosurfactants or other growth by-products in the broth are further separated and purified from the broth.

[0098] In other embodiments, the composition (yeast, broth, or yeast and broth) can be placed in a container of appropriate size, for example, considering the intended use, the expected method of application, the size of the fermentation tank, and any mode of transportation from the microbial growth facility to the place of use. Thus, the container in which the yeast-based composition is placed can be, for example, from 1 gallon to 2,000 gallons or more. In other embodiments, the container is 2 gallons, 5 gallons, 25 gallons, 250 gallons, or more.

[0099] In certain embodiments, the compositions of the present invention have advantages over, for example, biosurfactant alone, including one or more of the following. High concentrations of mannoproteins as part of the outer surface of the yeast cell wall (mannoproteins are very effective biosurfactants), the presence of the biopolymer beta-glucan (an emulsifier) in the yeast cell wall, and the presence of biosurfactants, metabolites, and solvents (such as lactic acid, ethanol, ethyl acetate, etc.) in the culture.

[0100] Other chemicals useful in accordance with the present invention include mannoproteins, beta-glucans, ethanol, lactic acid, and other metabolites having, for example, bioemulsifying properties and surface / interface tension reducing properties.

[0101] In certain embodiments, the microbial culture and / or growth by-product composition can be added to concrete. These compositions can be added during the initial mixing of the concrete components, or the microbial culture and / or growth by-product composition can be added after the initial concrete application, i.e., during or after the curing process.

[0102] Concrete has three basic components: water, cement, and aggregates. For example, aggregates such as sand, stone, rock, gravel, recycled concrete, glass, or blast furnace slag constitute most of the concrete, which forms the basic structure of the concrete. In certain embodiments, the aggregate can be of one type, such as sand, or a combination of different aggregates, such as sand, recycled concrete, and glass, for example. Cement is used as an adhesive to bind the aggregates together. Water is added to make the mixture a homogeneous, workable composition. This concrete mixture can be made in various shapes and compositions based on its intended use.

[0103] Various types of admixtures are added to the concrete mixture. These include, for example, accelerators, binders, corrosion inhibitors, air-entraining agents, crystalline admixtures, pigments, plasticizers, superplasticizers, pumping aids, retarders, water reducers, shrinkage reducing agents, hydration control admixtures, alkali-silica reaction inhibitors, moisture protection admixtures, permeability reducing admixtures, gas-forming admixtures, non-segregating admixtures, foaming admixtures, and / or workability admixtures.

[0104] In certain embodiments, the accelerator is used in or with a microbial culture and / or growth by-product composition. Calcium chloride, calcium nitrate, calcium nitrite, calcium formate, calcium chloride, triethanolamine, and sodium thiocyanate can be used in the concrete composition, but they often cause corrosion problems and require the use of corrosion inhibitors.

[0105] In certain embodiments, the corrosion inhibitor is used in or with a microbial culture and / or growth by-product composition. Examples of corrosion inhibitors include amino alcohols such as 2-aminomethylpropanol, benzotriazoles such as diethylethanolamine benzotriazole and methylbenzotriazole, calcium nitrite, and sodium monofluorophosphate.

[0106] Microbial cultures and / or growth by-products are applied together with a composition that promotes their adhesion to the surface to be treated. The adhesion promoter may be a component of the microbial culture and / or growth by-products, or may be applied continuously together with a yeast-based product. Binders, adhesives, resins, and epoxies are components used in concrete that can bond concrete to existing structures or bond additional chemicals to the surface of concrete. These binders assist in adhering fresh concrete to hardened concrete structures. Binders that can be used in the present disclosure include polyvinyl chloride, polyvinyl acetate, acrylic, and butadiene-styrene copolymers.

[0107] In certain embodiments, an air entraining agent can be used in or with the microbial culture and / or growth by-product composition. Examples of air entraining agents include salts of wood resins, synthetic detergents, salts of sulfonated lignin, salts of petroleum acids, salts of proteinaceous substances, fatty acids and resin acids and their salts, alkylbenzene sulfonates, and salts of sulfonated hydrocarbons.

[0108] In certain embodiments, a crystalline admixture is used in or with a microbial culture and / or growth by-product composition that includes, for example, latex and calcium stearate.

[0109] In certain embodiments, the microbial cultures and / or growth by - product compositions of the present invention contain pigments or dyes, which can impart color to paints or other coatings, but can also, for example, protect surfaces or objects from UV light. The pigments or dyes can be natural, synthetic, inorganic, or organic. The pigments or dyes can be selected, for example, from titanium dioxide, zinc oxide, zinc yellow, yellow dyes, benzidine yellow, chromium oxide green, phthalocyanine green, phthalocyanine blue, ultramarine blue, vermilion, pigment brown 6, red 170, dioxazine violet, carbon black, iron(II) oxide, quartz sand (SiO2), talc, barite (BaSO4), kaolin clay, and limestone (CaCO3).

[0110] In certain embodiments, plasticizers, superplasticizers, workability admixtures, and water - reducing admixtures can be added to or used in combination with the microbial cultures and / or growth by - product compositions. Some examples of chemical plasticizers include lignosulfates, polyglucoesters, carbohydrates, and hydroxylated carboxylic acids. There are also superplasticizers, such as modified lignosulfates, which are superior to plasticizers in the ability to enable the workability of concrete and contain sulfonated naphthalene formaldehyde.

[0111] In certain embodiments, pumping aids are added to or used in combination with the microbial cultures and / or growth by - product compositions. Pumping aids include organic and synthetic polymers, organic flocculants, organic emulsions of paraffin, coal tar asphalt, acrylics, bentonite and pyrogenic silica, and hydrated lime.

[0112] In certain embodiments, retarders and hydration - control admixtures are added to or used in combination with the microbial cultures and / or growth by - product compositions. Retarders include lignin, borax, sugars, tartaric acid, and salts.

[0113] In certain embodiments, the shrinkage reducing agent is added to, or used in combination with, a microbial culture and / or growth by-product composition, such as, for example, polyoxyalkylene alkyl ether and propylene glycol.

[0114] The alkali-silica reaction inhibitor can be used in or with a microbial culture and / or growth by-product composition. Examples of the alkali-silica reaction inhibitor include barium salts, lithium nitrate, lithium carbonate, and lithium hydroxide.

[0115] The moisture-proof or waterproof admixture can be used in or with a microbial culture and / or growth by-product composition. Examples of chemicals that can be used for moisture-proof concrete include, for example, calcium stearate or ammonium soap, calcium oleate or ammonium soap, butyl stearate, and various petroleum products. Waterproofing materials are also applied to the outer surface of the concrete. Some of the materials that can be used for waterproof concrete include rubberized asphalt coating, bentonite, rubber-based coating, and urethane coating.

[0116] In certain embodiments, the gas-forming admixture is added to, or used in combination with, a microbial culture and / or growth by-product composition. The most common gas-forming admixtures are, for example, aluminum powder, activated carbon, and hydrogen peroxide.

[0117] In certain embodiments, the non-separating admixture is added to, or used in combination with, a microbial culture and / or growth by-product composition. Cellulose and acrylic polymer are examples of substances that can be used in the non-separating admixture.

[0118] In certain embodiments, the foaming admixture is added to and / or used in combination with a microbial culture and / or growth by - product composition that includes a surfactant and a hydrolyzed protein. In certain embodiments, the concrete composition contains microbial growth by - products at a concentration of about 0.001% to about 50%, about 0.01% to about 10%, about 0.05% to about 1%, or about 0.1% to about 0.5% by weight or volume.

[0119] In certain embodiments, the composition applied to the surface of the concrete contains microbial growth by - products at a concentration of about 0.001% to about 50%, about 0.01% to about 10%, about 0.01% to about 1%, or about 0.1% to about 0.5% by weight or volume of the composition.

[0120] In certain embodiments, the microbial cells are present, for example, at a concentration of at least 1×10 4 、1×10 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 、1×10 10 、or 1×10 11 or more cells per milliliter in the composition.

[0121] Advantageously, according to the present invention, the microbial culture and / or growth by - products may include the broth in which the microorganisms have grown. The product may be, for example, at least 1 wt%, 5 wt%, 10 wt%, 25 wt%, 50 wt%, 75 wt%, or 100 wt% broth. The amount of biomass in the product is, for example, any of 0 wt% to 100 wt% including all percentages therebetween.

[0122] Optionally, the product can be stored before use. The storage time is preferably short. Thus, the storage time is less than 60 days, less than 45 days, less than 30 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 5 days, less than 3 days, less than 2 days, less than 1 day, or less than 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at a low temperature, such as less than 20 °C, 15 °C, 10 °C, or 5 °C. On the other hand, the biosurfactant composition can typically be stored at ambient temperature.

[0123] The compositions or methods described herein can be combined with one or more of the other compositions and methods described herein.

[0124] Other features and advantages of the present invention will become apparent from the following description of the preferred embodiments and from the claims. All references cited herein are hereby incorporated by reference.

[0125] Use of Microorganisms in Concrete and Their Growth By-Products The use of the compositions according to the invention in concrete provides various advantages. For use in a concrete mixture, the microbial culture and / or growth by-products can be added during the initial mixing of the various components of the concrete, including water, cement, aggregates, and any admixtures.

[0126] Both acidic and lactone sophorolipids can be used. The mixture between the two types of sophorolipids can be adjusted based on the desired properties for each product application. The ratio of sophorolipids to other components including yeast cells can be varied for each application. Sophorolipids can be purified from the organisms that produced the chemical substances and / or growth media. Alternatively, sophorolipids can be used in an unpurified form. The organisms used to produce the biosurfactant can be removed from the composition, while other components of the growth media and other by-products produced by the organisms can be left in the mixture added to the concrete composition.

[0127] As used herein, to “apply” a composition or product means to bring the composition or product into contact with a target, site or material such that the composition or product affects the target, site or material. This effect is, for example, due to microbial growth and / or the action of biosurfactants and other growth by-products. For example, a microbe-based composition or product can be added to a concrete mixture in liquid or dry form by injection, spraying, mixing, diffusion, and / or injection in a concrete batch plant and / or at the location where the concrete product is used.

[0128] When a microbial culture and / or growth by-product composition is applied to concrete during or after curing, it can be applied, for example, by spraying using a spray bottle or a pressurized spray device. The composition can also be applied using a cloth or a brush, where the composition is rubbed, spread, or brushed onto the surface. Further, the composition can be applied to a surface by dipping, submerging, or immersing the surface into a container containing the composition.

[0129] In one embodiment, a material and / or surface can be immersed in the composition. For example, the immersion can be carried out for at least 5 seconds, 30 seconds, 1 minute, 30 minutes, 60 minutes, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours, or more, as needed.

[0130] In certain embodiments, the microbial culture and / or growth by-products can improve concrete by increasing the strength of the concrete, reducing the amount of water used in the concrete composition, increasing the workability of the concrete, reducing the porosity of the concrete, altering the setting rate of the concrete, and / or increasing the durability of the concrete against biological and abiotic stresses.

[0131] Concrete composition Microbial cultures and / or growth by - product compositions can be used in common types of concrete / cement used in construction, such as Portland cement. Further, microbial cultures and / or growth by - product compositions can be used in asphalt concrete, which is often used in roads, parking lots, airports, and dams. Permeable concrete, nano - concrete, microbial concrete, and polymer concrete are all types of concrete in which microbial cultures and / or growth by - products can be used as components of the mixture.

[0132] Furthermore, biosurfactants can be used in coatings applied to concrete. Generally, concrete is coated to seal it from surface damage, corrosion, and contamination by preventing penetration of water, salts, and / or air. In certain embodiments, the microbial culture and / or growth by - products cover only the surface, while in other embodiments, the microbial culture and / or growth by - products penetrate the concrete and remain on the surface.

[0133] Improvement of Concrete Strength The strength of concrete typically varies inversely with the amount of water in the concrete mixture. A 1% decrease in the water concentration in concrete results in approximately a 5% increase in the strength of the concrete. However, in some cases, a decrease in the water content reduces the workability and setting time of the concrete.

[0134] According to the present invention, the addition of microbial cultures and / or growth by - products, preferably sophorolipids and W. anomalus can enhance the workability of concrete without sacrificing strength. To maintain the workability of concrete while increasing its strength, plasticizers and superplasticizers are currently used in concrete mixtures.

[0135] The strength of concrete can be improved by combining various methods or using them alone. The methods include, but are not limited to, reducing the amount of water in the concrete composition and reducing the porosity of the concrete. When water evaporates from the concrete during the curing process, voids are formed in the concrete. Therefore, the smaller the amount of voids, the stronger the concrete. These remaining voids determine the porosity of the concrete. Using microbial cultures and / or growth by-products to reduce the amount of water in the initial concrete mixture also reduces the porosity of the concrete. The porosity of the concrete is determined by the concentration of the microbial cultures and / or growth by-products added to the concrete, and it is possible to maintain the workability of the concrete while increasing its strength compared to a concrete mixture without microbial cultures and / or growth by-products.

[0136] In certain embodiments, adding sophorolipids to the concrete mixture at a concentration of about 0.01% to about 0.5% and adding yeast cell fermentation waste at a concentration of about 0.1% to about 0.5% can significantly increase the strength of the concrete. Yeast cell fermentation waste is the result of live or dead yeast cells. Therefore, yeast cells can be added at a concentration of about 0.1 g / l to about 100 g / l. Adding yeast cells, sophorolipids, and / or other growth by-products can also reduce water consumption by about 1% to about 50%, about 10% to about 25%, or about 15% to about 20%. This optional reduction in water can also change the curing time of the concrete.

[0137] Change in the curing time of concrete By adding a microbial culture and / or a growth by - product composition to a concrete mixture, the setting time can be changed. The concentration of water in the concrete determines the length of setting (when all other variables affecting evaporation are equal). When the water concentration is low, evaporation is fast and the setting time is short, while when the water concentration is high, water evaporation is slow and the setting time is long. To list the advantages, in certain embodiments, the addition of the composition of the present invention to the concrete mixture can reduce the amount of water required to keep the concrete workable. Thus, in some embodiments, the setting time of the concrete can be shortened.

[0138] Alternatively, in certain embodiments, by adding a microbial culture and / or a growth by - product composition to a concrete mixture, the setting time can be extended while maintaining or even increasing the concrete strength. Concrete is plastic over a long period.

[0139] Increasing the resistance of concrete to biological and abiotic stresses In certain embodiments, the microbial culture and / or the growth by - product composition is added to the concrete mixture or applied to the solidified surface of the concrete to enhance resistance to environmental stresses. The stresses include biological stresses such as bacteria and barnacles, high and low temperatures, freeze - thaw cycles, salts, high or low pressures, wind, sedimentation, waves, water, UV radiation, impurities in the concrete mixture or concrete coating, hydrostatic pressure, abrasion (such as sandstorms), and movement of the concrete, for example, non - biological stresses including the transportation of solidified concrete slabs by trucks.

[0140] In certain embodiments, concrete can be made frost-resistant by the addition of a microbial culture and / or growth by-products. Frost resistance refers to the ability of concrete to maintain its intended use even when used at temperatures below 0°C. Frost resistance can be achieved by preventing the penetration of moisture or humidity into the concrete, thereby preventing the formation of ice within the concrete and the potential for the concrete to break apart. Further, the microbial culture and / or growth by-products can enable the concrete to withstand concrete damage resulting from freezing at temperatures of about -30 o °C, about -15 o °C, or about -5 o °C or lower.

[0141] In certain embodiments, the microbial culture and / or growth by-product composition increases the lifespan of the concrete by preventing fouling by biological or abiotic substances. The present invention can be used to prevent the occurrence of deposits.

[0142] In certain embodiments, the microbial culture and / or growth by-product composition seals the concrete from penetration by water, salts, and / or air. The biosurfactant composition can remain on the surface of the concrete. Additionally, the composition can penetrate the concrete. The penetration can reach a depth of about 0.1% to about 1%, about 0.5% to about 5%, about 1% to about 10%, about 5% to about 50%, or 100% of the total depth of the concrete.

[0143] In certain embodiments, the microbial culture and / or growth by-product composition can prevent the concrete from coming into direct contact with abrasive elements such as waves and wind.

[0144] Evaluation of Concrete Various tests can be performed to identify and evaluate various properties of a concrete composition or a concrete coating. These tests can be used to verify that the concrete meets the structural requirements. Alternatively, these tests can be used to determine the optimal concentration of various admixtures to create the desired properties of the concrete.

[0145] Absorption test Testing the moisture level in concrete is a common method for determining the water resistance properties of concrete samples. One common absorption test is the one used in BS1881-122:2011.

[0146] The British Standard (BS) 1881-122:2011 and BS1881-122:1983 tests measure the amount of water that penetrates into concrete samples when immersed in water. These tests, along with variations of the tests, are known to those skilled in the art. In these two tests, various items are required. The test specimens are weighed using weights, cores with a diameter of approximately 75 mm are cut using a coring machine, a drying oven is used at a temperature of approximately 105°C, a tank with clean water maintained at a temperature of approximately 20°C and a depth of at least 125 mm, and a dried airtight container used as a desiccator with a volume sufficient to hold the three samples being tested are used. Each sample is processed according to tests recognized in the art for water permeability.

[0147] Rapid chloride permeability test The rapid chloride permeability test evaluates the ability of concrete to resist chloride ion penetration by monitoring the amount of charge passing through a cylindrical sample. An example of this test is ASTM C1202. In this test, charge is passed through the concrete for 6 hours. The total charge passing through the concrete is related to the ability of the test specimen to resist chloride ion penetration. A low charge level passing through indicates high resistance. The chloride permeability test is well known to those skilled in the art.

[0148] Water permeability test The water permeability test determines the depth of water penetration into a concrete sample when it is exposed to hydrostatic pressure. Two examples of water penetration tests, BS (European Standard) EN12390-8 and DIN1048 Part 5, test the depth of water penetration when a concrete sample is subjected to a hydrostatic pressure of 0.5 MPa for 3 days. The concrete sample is cast, cured for 28 days, and then placed in the testing apparatus. The sample holder is open at both ends, and one end is exposed to the hydrostatic pressure. After 3 days, the sample is removed from the testing apparatus, split vertically in half, and the maximum depth of water penetration is measured.

[0149] This test is useful for testing concrete used in structures such as basements, tunnels, dams, and water storage tanks, as it measures the water penetration into concrete under hydrostatic pressure. If a concrete structure is not intended to be subjected to hydrostatic pressure during its lifetime, this water permeability test can be combined with an absorption test.

[0150] Compressive Strength Test The compressive strength test checks the ability of concrete to function properly under an applied static or dynamic load. Some of the standard tests are ASTM C31, ASTM C39, ASTM C192, AASHTO T-22, AASHTO T-23, AASHTO T-126, EN 1290-1, EN 12390-2, EN 12390-3, EN 12390-4, and EN 12504-1.

[0151] This test may be used to determine the size or structural components necessary to meet the desired load, quality control, approval of the design mixture, and acceptability. The test can be carried out on-site or at the manufacturing location of the concrete application.

[0152] To measure the compressive strength, a cylindrical or cubic concrete specimen is placed in a compression testing machine under a constant loading rate, and the breaking load (the load at which concrete failure is measured) is measured. The strength is calculated by dividing the breaking load by the cross-sectional area of the specimen perpendicular to the load direction. The unit of strength is psi, kg / cm 2 , or MPa, among various pressure units.

[0153] The final test results are derived from the average strength of several specimens tested at the same curing age, which are generally specified to be cast from the same sample and have a fully designed capacity at 28 days (28-day strength). Three consecutive test results must meet or exceed the specified strength, and the test must not be lower than a specific percentage of the specified strength.

[0154] The equipment required in addition to the compression testing machine is a mold, a mixer, a testing device, and a sample curing device.

[0155] Tensile Strength Test The tensile strength test is to determine the resistance of concrete to failure when a tensile force is applied. Some of the standards for this test are ASTM C31, ASTM C78, ASTM C192, AASHTO T-23, AASHTO T-97, AASHTO T-126, EN12390-5, EN1339, EN1340, and EN1521.

[0156] Concrete is not usually designed for applications that require resistance to direct tensile loads because the tensile strength of concrete is typically 10 - 15% of its compressive strength. Some types of tensile strength tests used to identify the characteristics of concrete include direct tensile force application tests, flexure tests, indirect split cylinder tests, and, rarely, uniaxial tensile tests.

[0157] The flexure test is a common test that involves applying a flexural load to an unreinforced concrete prism or beam using symmetric two - point or three - point loading. The concrete beam is bent under the load, and the tensile strength and / or modulus of rupture are calculated using the load at the failure point when the beam fails. In the split - cylinder test, a cylindrical test specimen is placed horizontally with its long axis between the platens of a compression machine, and a load is incrementally applied to the side of the test specimen until it fails by splitting the cylinder along its axis.

[0158] The necessary equipment includes molds, mixers, testing devices, and sample curing devices. For the flexure test, a flexure beam frame or flexure platens are installed on a compression machine. In the split - cylinder test, a test fixture and wooden pieces are required to apply the splitting load.

[0159] Density Test There are various tests to determine the density of concrete, mainly by the buoyancy balance method of weighing a known volume under both dry and immersed conditions, and the yield bucket method of placing a fresh concrete sample in a metal container of known volume. The fresh concrete is compressed in the container, filled to the top, the top is scraped level, and then weighed. The density is determined by simple weight - volume calculations. Standard tests that can be used are ASTM C29, ASTM C138, AASHTO T - 19, AASHTO T - 121, EN 12390 - 7, and EN 1097 - 3.

[0160] The necessary equipment includes molds, mixers, a buoyancy balance and frame, scales, tamping rods, punching plates, and sample curing devices.

[0161] High-density concrete can be used when casting pre-tensioned reinforcement elements such as tensioned cables or reinforcing bars. The concrete is poured around the pre-tensioned cable to provide additional bond strength between the bar and the concrete. Once the concrete has hardened, the bar is released, compressing the concrete element. This is used for high-strength elements such as bridge girders and pre-tensioned floor slabs. Low-density concrete and air-entrained concrete are used for on-grade floor slabs to improve performance in adverse weather conditions. Concrete density varies depending on the density of the components, including the presence of biosurfactants, aggregates, cement, and its air content.

[0162] Slump test The concrete slump test is used to measure the consistency, workability, and ease of flow of fresh concrete. Generally, the test indicates the water-to-cement ratio, and a high water content indicates a high slump value. Slump is an indicator of the compressive strength of hardened concrete. Generally, in standard-weight concrete, the higher the water content, the lower the strength. However, admixtures containing biosurfactants affect the slump value by changing the water required for the necessary fluidity for the required cement ratio. Standard tests that can be used are ASTM C143, AASHTO T-119, and EN 12350-2.

[0163] The test is performed using a conical mold filled with fresh concrete, called a slump cone or Abrams cone of standardized dimensions. When the cone is removed, the fresh concrete settles vertically, and the slump value is the vertical settlement from the original height, i.e., the measured value of the slump. The required equipment is a metal slump cone, a tamping rod, a slump cone base, a tape measure, a mold, a mixer, a testing apparatus, and a sample curing apparatus.

[0164] High-flow concrete is used for casting concrete in high-strength steel formwork in order to ensure that a proper and uniform mixture is reliably distributed throughout the reinforced concrete element. Therefore, the main purpose of measuring the slump is to achieve acceptable workability.

[0165] Air content test The air content test is used to determine the amount of air in the concrete. The test usually uses the pressure method. The concrete is placed in a container of known volume and the top is washed away. This method is based on Boyle's law, and the air column in the voids is proportional to the applied pressure. Pressure is applied to the sealed test container by connecting a separate air chamber equipped with a pump. With the valve closed, the chamber is pressurized to the calibrated operating pressure and the pressure gauge is deflated. When the valve is opened, the air in the concrete expands into the test chamber and the gauge indicates a reading in units of air content. The necessary equipment is an air entrainment meter, a strike-off bar, a rubber mallet, a mixer, a testing device, and a sample curing device. The standard tests used are ASTM C231, AASHTO T-152, and EN 12350-7.

[0166] In locations where the concrete is exposed to cycles of freezing and thawing, a high level of air entrainment in the concrete is often required. Water expands when it freezes, which generates internal forces that exceed the bond or tensile strength of the concrete element and cause cracking. Air in the form of very small bubbles acts as a reservoir where water accumulates and expands, creating void spaces within the concrete that relieve the internal pressure during the freezing cycle and protect the concrete. The air is uniformly distributed throughout the concrete mixture using a mixing blade. Additives are used during the mixing process to stabilize the bubbles of entrained air and remain after the concrete has hardened.

[0167] Concrete test hammer Use a test hammer to determine the strength of in-situ concrete. A concrete test hammer is also known as a rebound hammer or a Schmidt hammer. The test hammer uses a spring-actuated mass that, when released, strikes the surface of a concrete sample with a designed amount of energy. The rebound distance after impact is measured. The hammer is held perpendicular to the surface being tested, and the rebound varies according to the hardness of the sampling point. This rebound measurement is converted to compressive strength using a conversion table. Different conversion tables are made to account for the orientation of the instrument. Such tables were developed by performing rebound tests on concrete samples before they were crushed under compression. The equipment required in addition to a compression testing machine are molds, mixers, testing equipment, and sample curing equipment.

[0168] This is not a standard test for determining compressive strength as outlined previously. However, the number of concrete test specimens to be compression tested is often too few to be considered compared to random testing. Also, in many cases, it is important to determine the compressive strength of aged concrete elements for the retrofit, modeling, and analysis of structures.

[0169] Ultrasonic pulse velocity test Ultrasonic concrete tests include measuring the homogeneity of concrete, determining the presence of voids, cracks, and other defects, assessing the deterioration of concrete caused by aging, fire, frost, or chemical attack, measuring layer thickness and elastic modulus, and determining concrete strength. To test these parameters, an ultrasonic pulse is passed through the concrete and the travel time is measured.

[0170] This test is performed on hardened concrete to evaluate its quality. When a high speed of ultrasonic pulse is observed, the concrete is homogeneous and uniform throughout with respect to density or the presence of defects. Surface and subsurface problems can be detected by this method.

[0171] Examples The present invention and many of its advantages will be more deeply understood from the following examples given by way of illustration. The following examples illustrate some of the methods, uses, embodiments, and variations of the present invention. The examples do not limit the present invention.

[0172] Example 1 - Sophorolipids and W. anomalus Increase in Concrete Compressive Strength by The combination of biosurfactant and yeast culture improves the strength of concrete. The strength of the samples was evaluated using compressive strength tests. Each sample contained Portland cement, sand, and water. Some samples further contained W. anomalus either lactone or acidic sophorolipids.

[0173] Under the following test conditions, 150 ml of water was added per 1 kg of QUIKRETE® without admixture to a conventional concrete mixture containing water and QUIKRETE®, and W. anomalus and sophorolipids were added. 1. Control: 0 g / l of W. anomalus and 0 ml / l of sophorolipids 2. Group 2: 10 g / l of W. anomalus and 0.05 ml / l of lactone sophorolipids 3. Group 3: 10 g / l of W. anomalus and 0.1 ml / l of lactone sophorolipids 4. Group 4: 10 g / l of W. anomalus and 0.3 ml / l of acidic sophorolipids 5. Group 5: 10 g / l of W. anomalus and 0.1 ml / l of acidic sophorolipids 6. Group 6: 10 g / l of W. anomalus and 0.05 ml / l of acidic sophorolipids

[0174] The concrete mixture with or without yeast and biosurfactant is poured into cylinders having a fixed diameter of 50 mm and a height of 100 mm. The concrete samples are dried at room temperature (22 °C) for 27 days.

[0175] To determine the compressive strength of each concrete sample, a TORBAL FB high-resolution precision force gauge was used. Each concrete sample cylinder was placed vertically on a stand with the gauge leveled and the tare weight removed. Compression was applied until cracks occurred in the cylinder and the force could no longer be measured. The maximum force obtained was recorded in Table 1. Each test condition was performed three times. The average maximum compressive force obtained was determined for each trial condition.

[0176]

Table 1

[0177] Based on the results shown in Table 1, the concrete mixture containing 10 g / l of W. anomalus and 0.05 ml / l of lactone sophorolipid had a compressive strength 21.4% higher than that of the control concrete without either yeast cells or sophorolipid.

Claims

**Claim 1**: A composition for improving the strength of concrete, comprising an inactive Starmerella bombicola or Wickerhamomyces anomalus yeast culture, lactone sophorolipid as a biosurfactant, and a concrete mixture. **Claim 2** The concrete mixture according to claim 1, wherein the concrete mixture comprises water, aggregates and cement, and the aggregates are selected from one or a combination of sand, gravel, stone, rock, blast furnace slag, glass, recycled concrete. **Claim 3** The composition according to claim 1, wherein the lactone sophorolipid is present in the composition at a concentration of about 0.001% to about 5%. **Claim 4** The composition according to claim 1, wherein the yeast culture is present in the composition at a concentration of about 0.1 g / l to about 100 g / l. **Claim 5** The composition according to claim 1, wherein the yeast culture comprises one or a combination of carbohydrates, polyols, lipids, glycolipids, esters or proteins. **Claim 6** The composition according to claim 1, further comprising a concrete admixture. **Claim 7** The concrete admixture is a) an accelerator, b) a binder, c) a corrosion inhibitor, d) an air entraining agent, e) a crystalline admixture, f) a pigment, g) a plasticizer, h) a superplasticizer, i) a pumping aid, j) a retarder, k) a water reducer, l) a shrinkage reducing agent, m) a hydration control admixture, n) an alkali-silica reaction inhibitor, o) a moisture protection admixture, p) a permeability reducing admixture, q) a gas forming admixture, r) a segregation preventing agent, s) a foaming mixture, and t) a workability admixture The composition according to claim 6, comprising at least one of the above. **Claim 8**: A method for improving the strength of concrete, comprising adding an inactive Starmerella bombicola or Wickerhamomyces anomalus yeast culture and lactone sophorolipid as a biosurfactant to the concrete. **Claim 9** The method according to claim 8, wherein the concrete comprises water, aggregates, and cement. **Claim 10** The method according to claim 9, wherein the concrete further comprises a concrete admixture. **Claim 11** The concrete admixture is a) an accelerator, b) a binder, c) a corrosion inhibitor, d) an air entraining agent, e) a crystalline admixture, f) a pigment, g) a plasticizer, h) a superplasticizer, i) a pumping aid, j) a retarder, k) a water reducer, l) a shrinkage reducing agent, m) a hydration control admixture, n) an alkali-silica reaction inhibitor, o) a moisture protection admixture p) A magnetic permeability reducing admixture, q) A gas forming admixture, r) A non-separating agent, s) A foaming mixture, and t) A workability admixture The method according to claim 10, selected from one or a combination thereof.

12. The method according to claim 9, wherein the aggregate is selected from one or a combination of sand, gravel, stone, rock, blast furnace slag, glass, and recycled concrete.

13. The method according to claim 8, wherein the yeast culture contains one of a combination of carbohydrates, polyols, lipids, glycolipids, esters, and proteins.

14. The method according to claim 8, wherein the lactone sophorolipid is present at a concentration of about 0.001% to about 50%.

15. The method according to claim 8, wherein the yeast culture is present in the composition at a concentration of about 0.1 g / l to about 100 g / l.

16. The method according to claim 8, wherein the concrete, lactone sophorolipid, and / or yeast culture are mixed together prior to application of the concrete.

17. The method according to claim 8, wherein the concrete hardens when the yeast culture and / or lactone sophorolipid are applied to the concrete.

18. The method according to claim 8, wherein the concrete is completely hardened when the yeast culture and / or lactone sophorolipid are applied to the concrete.

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