WO2019222168A1 - Production and preservation of bacillus reference culture for generating standardized and reliable inocula - Google Patents

Abstract

The subject invention provides methods of producing and preserving stable, standardized reference cultures for use in inoculating fermentation reactors used in research labs and other commercial applications. In specific embodiments, the reference cultures comprise bacterial spores stabilized using, for example, 0.1 to 0.5% phenol, that can be stored for a period of multiple days, weeks, months or even years.

Description

PRODUCTION AND PRESERVATION OF BACILLUS REFERENCE CULTURE FOR

GENERATING STANDARDIZED AND RELIABLE INOCULA

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application Serial No. 62/671,111, filed May 14, 2018, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cultivation of microorganisms such as bacteria, yeasts and fungi is important for the production of a wide variety of useful bio-preparations. Microorganisms play crucial roles in, for example, the food industry, pharmaceuticals, agriculture, mining, environmental remediation, and waste management.

Two principle forms of cultivation of microorganisms exist for producing bacteria, yeasts and fungi, which include submerged cultivation and surface cultivation. Both cultivation methods require a nutrient medium for the growth of the microorganisms. The nutrient medium, which can either be in a liquid or a solid form, typically includes a carbon source, a nitrogen source, salts and appropriate additional nutrients and microelements. The pH and oxygen levels are maintained at values suitable for a given microorganism.

Often, inoculum cultures, or inocula, are used to prepare a scaled population of microorganisms in a culture medium. To obtain inocula, microbiology laboratories routinely use standardized reference cultures, which can be tapped for a desired quantity of microorganisms. Reference cultures are generally prepared by diluting a culture of microorganisms to obtain a fresh cell suspension that contains an estimated number of colony-forming units per milliliter (CFU/mL). Aliquots of this culture can then be used, for example, to inoculate each of a series of repeated experiments.

As a first step, inoculum is taken from a working stock culture or a reference culture, to initiate growth in a suitable nutrient medium. For example, bacterial vegetative cells and spores can be added to fermentation broth. Inoculum development is usually performed in a stepwise fashion to increase the volume of culture to the desired level. This stepwise process can lead to variability in yields and productivity, which in turn can lead to inefficient cultivation and substantial time spent preparing inocula.

Additionally, the accuracy with which the number of CFU/mL can be determined often varies greatly due to the extrapolation of small measurement errors during dilution, which is compounded by the biological variability of the sample and the potential for unwanted cell growth. As such, using subsequently prepared, fresh reference cultures naturally increases the potential for false or invalid results, as it is difficult to consistently determine the number of CFU/mL for each single inoculum in a series of experiments. Furthermore, because of the multitude of stages of cultivation involved, there is a high probability for mutations to occur in the culture, leading to unpredictable future genotypes.

One solution to the instability and inconsistency between inocula is to dry microorganisms in a reference culture, which involves removing water from the cells. Dry form microbes can be preserved for extended periods of time, without changes in cell concentration or genetic makeup, and without contamination from undesirable microbial strains. For example, bacterial spores, such as those used in the food and supplement industry for producing probiotics, are often preserved in spray- dried or freeze-dried form. If performed correctly, drying of bacteria can help eliminate the need to cultivate inoculum cultures repeatedly, and can keep the bacteria in a desired metabolic state to prolong shelf life.

Conventional freeze-drying and spray-drying methods, however, are inconsistent, and often provide only limited success in delivery of metabolically viable cells at high densities. This is because of, for example, injury to the cells due to rapid variations in drying parameters. For example, rapid changes in temperature, amount of time spent in industrial dryers, as well as the spray nozzle type and pressure, air flow positioning, and other parameters, can affect the end viability of the cells. The result is often a reference culture with low survival and low preservation of metabolic capabilities, as well as low cell density. The long“lag” phases that necessarily ensue in commercial production can correspond with significant economic loss.

At the present time, bacteria are utilized in a wide range of commercial applications. Biosurfactant-producing bacteria, such as Bacillus, are used in enhanced crude oil recovery and as biopesticides for agricultural crops. Lactic acid bacteria cultures are used to produce cheese, yogurt, and other dairy products. Lactobacillus acidophilu and Bifidobacteria are extensively used as probiotics. Genetically altered bacteria, such as E. coli, are widely used as expression hosts for a variety of proteins and other products. Unfortunately, broader applications of bacteria and other cell cultures are limited due to deficiencies in conventional preservation methods and conventional production of reliable, stable reference cultures from which consistent inocula can be obtained.

Currently, providing inocula containing a relatively precise and consistent number of microorganisms with a reproducible amount of variation is a complex process. Furthermore, the difficulties that can come with preparation and long-term storage of reference cultures can lead to high costs and potential for contamination and loss of microbial activity.

Thus, there is a need for inexpensive and straightforward methods for producing and preserving stable, reliable reference cultures that can be used repeatedly for inoculating fermentation reactors of any scale.

SUMMARY OF THE INVENTION

The present invention provides methods for producing and preserving microorganisms that can be used in research laboratories and in industries such as oil and gas, agriculture, bioremediation, nutritional supplements, aquaculture, human and animal health and many others. Specifically, the subject invention provides methods and materials for efficient cultivation and preservation of a batch of reference culture that can be used to inoculate a cultivation reactor. Advantageously, the methods allow for preservation of the reference culture without resorting to freeze-drying or spray-drying, which helps to insure consistency, stability and reliability of the reference culture over time.

In specific embodiments, the methods provide for production of a bacterial reference culture that can be preserved and stored for an extended period of time, for example a period of months or even several years or more. Additionally, the subject invention provides methods of producing standardized, reliable inocula from the reference culture and methods of inoculating a cultivation reactor for scaled-up production of microorganisms and microbe-based products.

The subject methods are inexpensive to implement, and can significantly decrease the amount of work, time and capital that must be spent producing cultures for research and/or commercial uses. The methods can help prevent the reference culture from developing mutations over time, thus providing for genetically consistent inocula for research and production. Furthermore, the present invention can simplify production and facilitate consistent repetition of scaled cultivation procedures.

Organisms that can be cultured using the subject invention can include, for example, yeasts, fungi, bacteria, archaea, protozoa and viruses. In preferred embodiments, the microorganisms are bacteria. Even more preferably, the microorganisms are spore-forming Bacillus species.

In one embodiment, the subject invention provides a method of producing a stable, reliable reference culture for, e.g., inoculating cultivation reactors, wherein the method comprises cultivating a microorganism in a first cultivation reactor; allowing the microorganism to sporulate; and adding phenol to the first reactor to stabilize the spores. As used herein,“stable” spores are spores that experience minimal to no germination and/or change in microbial concentration (e.g., less than +/- 10% change), while maintaining the ability to germinate and resume metabolic activity in the future.

Advantageously, the culture can be stored in the first reactor in this stable form, at ambient temperatures (e.g., about 20°C to 25°C) or refrigerated at 0°C to 20°C , or 0°C to 4°C, for an extended period of time, for example, for one month or even one year or more. Furthermore, the stabilized spore culture can be stored without being contaminated by outside microorganisms.

In one embodiment, the reference culture can be used to inoculate a second cultivation reactor. For example, in one embodiment, a method of producing a microbial culture and/or a microbial growth by-product is provided, wherein an aliquot of the subject reference culture is removed from the first cultivation reactor and placed into a second cultivation reactor having nutrient medium therein.

The aliquot can have a volume of, for example, 0.1 mΐ to 10 liters. In preferred embodiments, the aliquot comprises stabilized microbial spores, e.g., at a concentration of 1% to 20%, or from 10% to 20% culture per unit volume. Advantageously, in certain embodiments, the subject methods allow for larger scale fermentation to be achieved with reduced time. The method can further comprise cultivating the aliquot of reference culture to a desired concentration according to standard procedures, depending on the type of reactor being used. Preferably, the nutrient medium in the second reactor does not comprise a spore stabilizer or preservative composition. In certain embodiments, however, germination enhancers can be added to the nutrient medium, such as, for example, L-alanine and/or manganese.

In one embodiment, a method is provided for preserving a reference culture comprising bacterial spores, wherein the method comprises adding phenol to the reactor where the culture was produced (e.g., the first reactor) in order to stabilize the spores. The stabilized spore culture can be stored for as long as 1 month, 6 months, or 1, 2, 3, 4, 5, or even 10 years, or longer without losing its stability and/or its efficacy. In one embodiment, the stabilized spore culture can be transferred into multiple separate flasks for ease of transport and storage.

In one embodiment, the subject invention provides methods of producing a surfactant, solvent, enzyme, and/or other metabolite, by cultivating an inoculant according to the methods of the subject invention under conditions appropriate for growth and metabolite production; and, optionally, purifying the metabolite.

Compositions produced according to the present invention can be used to inoculate large- scale fermentation systems for use in a wide variety of applications, including, for example, research laboratories, as well as the oil and gas industry, agriculture, human and animal health, food preservation, bioremediation, pharmaceuticals, cosmetics, aquaculture, horticulture, waste removal, and countless others.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for producing and preserving microorganisms that can be used in research laboratories and in industries such as oil and gas, agriculture, bioremediation, aquaculture, human and animal health and many others. Specifically, the subject invention provides methods and materials for efficient cultivation and preservation of a batch of reference culture that can be used to inoculate a cultivation reactor. Advantageously, the methods allow for preservation of the reference culture without resorting to freeze-drying or spray-drying, which can help to insure consistency, stability and reliability of the reference culture over time.

Selected Definitions

As used herein, reference to a“microbe-based composition” means a composition that comprises components that were produced as the result of the growth of microorganisms or other cell cultures. Thus, the microbe-based composition may comprise the microbes themselves and/or by products of microbial growth. The microbes may be in a vegetative state, in spore form, in mycelial form, in any other form of propagule, or a mixture of these. The microbes may be planktonic or in a biofilm form, or a mixture of both. The by-products of growth may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. The microbes may be intact or lysed. The microbes may be present, with medium in which they were grown, in the microbe-based composition, at, for example, a concentration of at least 1 x 10⁴, 1 x 10³, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10^u, 1 x 10¹² or more CFU/milliliter of the composition.

The subject invention further provides“microbe-based products,” which are products that are to be applied in practice to achieve a desired result. The microbe-based product can be simply the microbe-based composition harvested from the microbe cultivation process. Alternatively, the microbe-based product may comprise further ingredients that have been added. These additional ingredients can include, for example, stabilizers, buffers, appropriate carriers, such as water, salt solutions, or any other appropriate carrier, added nutrients to support further microbial growth, non nutrient growth enhancers, and/or agents that facilitate tracking of the microbes and/or the composition in the environment to which it is applied. The microbe-based product may also comprise mixtures of microbe-based compositions. The microbe-based product may also comprise one or more components of a microbe-based composition that have been processed in some way such as, but not limited to, filtering, centrifugation, lysing, drying, purification and the like.

As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or organic compound such as a small molecule (e.g., those described below), is substantially free of other compounds, such as cellular material, with which it is associated in nature. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. A purified or isolated polypeptide is free of the amino acids or sequences that flank it in its naturally-occurring state. A purified or isolated microbial strain means is removed from the environment in which it exists in nature; thus, the isolated strain may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with a carrier.

In certain embodiments, purified compounds are at least 60% by weight the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that 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 appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis.

A“metabolite” refers to any substance produced by metabolism or a substance necessary for taking part in a particular metabolic process. Examples of metabolites include, but are not limited to, enzymes, acids, solvents, alcohols, proteins, vitamins, minerals, microelements, amino acids, biopolymers, biosurfactants, and carbohydrates. The term“reference culture” refers to a standard or control batch of microbial culture, which is used as a source of culture for inoculating cultivation reactors. The microorganisms in a reference culture are preferably standardized, or genetically identical.

The term“inoculum” (plural“inocula”) can be encompassed within the term“microbe-based product.” As used herein, inoculum means a microbe-based product that can be used, for example, as a seed culture to inoculate a larger scale fermentation system or process. The inoculum can be an aliquot of a reference culture, which can be used to produce microorganisms and/or their growth by products, on any scale. The inocula of the present invention preferably comprise culture in an amount of approximately 1 to 50% by volume, or 5 to 40%, or 10 to 30%, or 15 to 20%.

As used herein,“on-site fermentation system” refers to a system used for producing microbe- based compositions and/or products at or near to the site of application of these microbe-based compositions and/or products. The on-site fermentation system can be, for example, less than 1 mile, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or 100 miles away from the site of application, or any number of miles within this range.

As used herein,“harvested” refers to removing some or all of the microbe-based composition from a growth vessel.

As used herein, the term“plurality” refers to any number or amount greater than one.

As used herein, the term“probiotic” refers to microorganisms, which, when administered in adequate amounts, confer a health benefit on the host. The probiotics may be available in foods and dietary supplements (for example through capsules, tablets, and powders). Non-limiting examples of common foods containing probiotics include dairy products such as yogurt, fermented and unfermented milk, smoothies, kefir, tea, kombucha, salad dressing, miso, tempeh, nutrition bars, and some juices and soy beverages. In preferred embodiments, the microorganisms are live or in spore form.

By“reduces” is meant a negative alteration of at least 0.001%, 0.1%, 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, or 100%. By“increase” is meant a positive alteration of at least 0.001%, 0.1%, 0.5%, 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

A“salt-tolerant” microorganism is capable of growing in a sodium chloride concentration of fifteen (15) percent or greater. In a specific embodiment,“salt-tolerant” refers to the ability to grow in 150 g/L or more of NaCl.

By“surfactant” is meant compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Surfactants act as, for example, detergents, wetting agents, emulsifiers, foaming agents, and dispersants. A“biosurfactant” is a surfactant produced by a living cell.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination of numbers, or subrange from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 as well as all intervening decimal values between the aforementioned 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” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of l to 50 may comprise l to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

The transitional term“comprising,” which is synonymous with“including,” or“containing,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By 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 a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Use of the term “comprising” contemplates other embodiments that“consist” or“consist essentially” of the recited component(s).

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms“a,” “an,” and“the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference.

Production and Preservation of Reference Culture

In specific embodiments, the subject invention provides methods of producing and preserving reference culture that can be stored for an extended period of time, for example a period of months or even several years or more, without losing stability and/or viability.

The subject methods are inexpensive to implement, and can significantly decrease the amount of work, capital and time that must be spent producing cultures for research and/or other uses. The method can help prevent the reference culture from developing mutations over time, thus providing for genetically consistent inocula for research and production. Furthermore, the present invention can simplify production and facilitate consistent repetition of cultivation procedures.

In specific embodiments, the subject invention provides a method of producing a stable, reliable reference culture for inoculating cultivation reactors, wherein the method comprises cultivating a microorganism in a first cultivation reactor; allowing the microorganism to sporulate; and adding phenol to the first reactor to stabilize the spores.

Advantageously, the culture can be kept and stored in the first reactor in this stable form, at ambient temperatures (e.g., about 20°C to 25°C) or refrigerated at 0°C to 20°C, or 0°C to 4°C, for an extended period of time, for example, for one month or even one year or more. Furthermore, the stabilized spores can stay in the reactor with minimal to no change in microbial concentration (e.g., less than +/- 10% change) and without loss of activity.

In one embodiment, the reference culture can comprise bacterial spores at a concentration of at least 1 x 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10^s, 10⁹, 10¹⁰, 10^{1 1} or 10¹² spores /ml or more.

Cultivation of the microorganism can be performed according to the methods described below. Preferably, the cultivation step is carried out until at least 80%, 85%, 90%, 95% or 100% of the microbial culture has sporulated.

Many bacterial species can form spores when, for example, certain nutrients in the environment become depleted, or the environment is otherwise not conducive to growth. Spores are formed, for example, in response to environmental stressors, within a“mother” cell compartment. Once the mother cell lyses, the resilient spores are released into the environment. Other than an initial, brief metabolic spurt, spores have little or no metabolic activity and are thus considered dormant. Certain environmental cues, such as temperature and nutrients, trigger the spores to begin germinating into vegetative cells once more.

After cultivation and sporulation of the microorganism, the method of producing a stable reference culture further comprises adding a spore stabilizing substance to the first cultivation reactor. Spore stabilizers, or preservatives, can include, but are not limited to, phenol, ethanol and isopropyl alcohol.

In preferred embodiments, the spore stabilizer is 0.001 to 5.0%, or 0.01 to 2.5%, or 0.1 to 0.5% v/v phenol. Advantageously, using phenol allows for stable preservation of spores, along with faster initiation of germination once it is desired.

In one embodiment, a method is provided for preserving a reference culture comprising bacterial spores, wherein the method comprises adding phenol to the reactor where the culture was produced in order to stabilize the spores. The stabilized spore culture that is produced can be stored for as long as 1 month, 6 months, or 1, 2, 3, 4, 5, or even 10 years, or longer without losing its stability and/or its efficacy. In one embodiment, the stabilized spore culture can be placed into one or more storage containers, such as, for example, plastic or glass tubes, vials, dishes, flasks or other standard laboratory containers for ease of transport and/or storage.

Advantageously, the methods of preserving a reference culture eliminate the need to dry the spores, and the temperature can be kept at ambient or slightly lower than ambient levels, for example, from 0°C to 25 °C, or from 4°C to 23 °C, or 20°C.

Use of Reference Culture for Inoculation

Compositions produced according to the present invention can be used to inoculate large- scale fermentation systems for use in a wide variety of applications, including, for example, laboratory research, as well as in industries such as the oil and gas industry, agriculture, human and animal health, bioremediation, food preservation, pharmaceuticals and cosmetics.

In one embodiment, the reference culture can be used to inoculate a second cultivation reactor. For example, in one embodiment, a method of producing a microbial culture and/or a microbial growth by-product is provided, wherein an aliquot of the subject reference culture is removed from the first cultivation reactor (or from a storage container, e.g., flask, in which it was stored) and placed into a second cultivation reactor having nutrient medium therein.

The aliquot can have a volume of, for example, from 0.1 mΐ up to 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 50 ml, 100 ml, 500 ml, 1 liter, 5 liters, or 10 liters.

In preferred embodiments, the aliquot comprises stabilized microbial spores, e.g., at a concentration of 1% to 20%, or from 10% to 20% culture per unit volume. Advantageously, in certain embodiments, the subject methods allow for larger scale fermentation to be achieved with reduced time.

Inoculation can be achieved using standard laboratory equipment and procedures, including, for example, by pouring, or if more precise inoculation is desired, using pipettes.

The method can further comprise cultivating the inoculum of reference culture to a desired concentration according to standard procedures, depending on the type of reactor being used. The inoculum can be cultured to a desired concentration using, for example, a shaker or drum mixer, or any other cultivation reactor. Preferably, the temperature of cultivation in the second cultivation reactor is between 10°C and 50°C, depending on the temperature of storage, as well as the microorganism of the culture (e.g., achieving a temperature increase of between 10°C and 25°C).

Preferably, the nutrient medium in the second reactor does not comprise a spore stabilizer or preservative composition. Thus, once the inoculum is placed into the nutrient medium, the amount of phenol (or other stabilizer) in the culture is significantly diluted compared to the concentration present in the reference culture. This promotes germination, thus reducing the time for total germination and cell growth to occur. In certain embodiments, however, germination enhancers can be added to the nutrient medium, such as, for example, L-alanine, manganese, L-va!ine, L-tyrosine, L-asparagine or any other known germination enhancer.

In one embodiment, the subject methods can further be used to produce a microbial growth by-product, such as, for example, a biosurfactant, solvent, enzyme, and/or other metabolite, by cultivating an inoculum of a microbe strain according to the methods of the subject invention under conditions appropriate for growth and metabolite production. In another embodiment, the method for producing microbial growth by-products may further comprise steps of concentrating and purifying the by-product of interest.

Advantageously, the subject invention reduces the capital and labor costs of producing microorganisms and their metabolites. Furthermore, the cultivation process of the subject invention reduces or eliminates the need to concentrate microbes or otherwise test and/or process the microbes after completing scaled-up cultivation. Even further, fermentation cycles can be shortened due to expedited germination turnover.

Methods of Cultivating Microorganisms

The subject invention provides methods of cultivating a stable reference culture, as well as methods of scaled-up cultivation of microbe-based compositions that are inoculated using aliquots of the reference culture. The reference cultures, and/or microbe-based compositions inoculated from the reference cultures, can be cultivated using fermentation methods known in the art, for example, through cultivation processes ranging from small to large scale. The cultivation process can be, for example, submerged cultivation, solid state fermentation (SSF), and/or a combination thereof.

The microbe growth vessel used according to the subject invention can be any enclosed fermenter or cultivation reactor for laboratory or industrial use. Preferably, the growth vessel is a simple cultivation reactor that does not require complicated operating procedures or materials.

The method can provide easy oxygenation of the growing culture with, for example, slow motion of air to remove low-oxygen containing air and introduction of oxygenated air. The oxygenated air may be ambient air supplemented periodically, such as daily.

In one embodiment, the vessel may optionally have functional controls/sensors or may be connected to functional controls/sensors to measure important factors in the cultivation process, such as pFI, oxygen, pressure, temperature, agitator shaft power, humidity, viscosity and/or microbial density and/or metabolite concentration.

In a further embodiment, the vessel may also be able to monitor the growth of microorganisms inside the vessel (e.g., measurement of cell number and growth phases). Alternatively, a daily sample may be taken from the vessel and subjected to enumeration by techniques known in the art, such as dilution plating technique. The method and equipment for cultivation of microorganisms and production of the microbial by-products can be performed in a batch process or a quasi-continuous process.

The microbes can be grown in planktonic form or as biofilm. In the case of biofilm, the vessel may have within it a substrate upon which the microbes can be grown in a biofilm state. The system may also have, for example, the capacity to apply stimuli (such as shear stress) that encourages and/or improves the biofilm growth characteristics.

In one embodiment, a mobile or portable bioreactor is used, which may be provided for on site production of a liquid batch culture for producing inocula including a suitable amount of a desired strain of microorganism. The amount of liquid culture produced can be, for example, 2 to 500 liters, 5 to 250 liters, 10 to 100 liters, 15 to 75 liters, 20 to 50 liters, or 35 to 40 liters.

In one embodiment, total sterilization of equipment and substrate used in the subject cultivation methods is not necessary. However, the equipment and substrate can optionally be sterilized. The cultivation equipment such as the reactor/vessel may be separated from, but connected to, a sterilizing unit, e.g., an autoclave. The cultivation equipment may also have a sterilizing unit that sterilizes in situ before starting the inoculation. Air can be sterilized by methods know in the art. For example, air can pass through at least one filter before being introduced into the vessel. In other embodiments, the medium may be pasteurized or, optionally, no heat at all added, where the use of low water activity and low pH may be exploited to control bacterial growth.

In one embodiment, the reactors and other equipment can be simply sanitized or decontaminated using, for example, UV light, detergents, bleach and/or hydrogen peroxide. In one embodiment, bleach and hydrogen peroxide in concentrated form can be diluted at the fermentation site before use. For example, the hydrogen peroxide can be provided in concentrated form and be diluted to formulate 1.0% to 3.0% hydrogen peroxide (by weight or volume). This can be done before or after a hot water rinse at, e.g., 80-90 °C to prevent contamination. The culture medium components (e.g., the carbon source, water, lipid source, micronutrients, etc.) can also be temperature decontaminated and/or hydrogen peroxide decontaminated (potentially followed by neutralizing the hydrogen peroxide using an acid such as HC1, H₂S0₄, etc.).

Advantageously, the fermentation vessel can also be self-sterilizing. For example, microorganisms chosen for cultivation can be strains known to produce antimicrobial metabolites or byproducts, such as biosurfactants. Thus, the microbe culture itself can provide control of unwanted microorganisms inside the device, simultaneously with cultivation of the desired microorganisms.

The method can comprise adding one or more antimicrobial substances to prevent contamination before, during or after cultivation (e.g., streptomycin, oxytetracycline, sophorolipid, and rhamnolipid).

Additionally, antifoaming agents may also be added to prevent the formation and/or accumulation of foam during cultivation and fermentation. In one embodiment, the cultivation can be supplemented with one or more organic and/or inorganic nitrogen sources. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride, as well as proteins, amino acids, yeast extracts, yeast autolysates, com peptone, casein hydrolysate, and soybean protein. These nitrogen sources may be used independently or in a combination of two or more.

The method can further comprise supplementing the cultivation with a carbon source. The carbon source is typically a carbohydrate, such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; fats and oils such as soybean oil, coconut oil, canola oil, rice bran oil, olive oil, corn oil, sesame oil, and/or linseed oil; etc. Other carbon sources can include one or more sugars such as xylose, galactose, sorbose, ribose, arbutin, raffmose erythritol, xylitol, gluconate, citrate, molasses, hydrolyzed starch, com syrup, and hydrolyzed cellulosic material including glucose. These carbon sources may be used independently or in a combination of two or more.

In one embodiment, growth factors and trace nutrients for microorganisms are included in the medium. This is particularly preferred when growing microbes that are incapable of producing all of the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium. Furthermore, sources of vitamins, essential amino acids, and microelements can be included, for example, in the form of flours or meals, such as com flour, or in the form of extracts, such as yeast extract, potato extract, beef extract, soybean extract, banana peel extract, and the like, or in purified forms. Amino acids such as, for example, those useful for biosynthesis of proteins, can also be included.

The method can comprise adding one or more lipid sources such as, for example, oils or fats of plant or animal origin that contain free fatty acids or their salts or their esters, including triglycerides. Examples of fatty acids include, but are not limited to, free and esterified fatty acids containing from 16 to 18 carbon atoms, hydrophobic carbon sources, palm oil, animal fats, coconut oil, oleic acid, soybean oil, sunflower oil, canola oil, stearic and palmitic acid.

The method can comprise adding one or more micronutrient sources, such as potassium, magnesium, calcium, zinc and manganese, preferably as salts; phosphorous, such as from phosphates; and other growth stimulating components. In one embodiment, inorganic salts may also be included. Usable inorganic salts can be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate (e.g., ferrous sulfate heptahydrate), iron chloride, manganese sulfate, manganese sulfate monohydrate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, calcium chloride, calcium carbonate, and/or sodium carbonate. These inorganic salts may be used independently or in a combination of two or more.

Each of the sources of nutrients can be provided in an individual package that can be added to the mixing apparatus at appropriate times during the cultivation process. Each of the packages can include several sub-packages that can be added at specific points ( e.g ., when culture, pH, and/or nutrient levels go above or below a specific concentration) or designated times (e.g., after 10 hours, 20 hours, 30 hours, 40 hours, etc.) during the cultivation process.

In one embodiment, the method for cultivation of microorganisms is carried out at about 5° to about 100° C, preferably, 15° to 60° C, more preferably, 20 to 50° C or 25 to 40 °C. In a further embodiment, the cultivation may be carried out continuously at a constant temperature. In another embodiment, the cultivation may be subject to changing temperatures.

A thermometer can be used to monitor temperature and the thermometer can be manual or automatic. An automatic thermometer can manage the heat and cooling sources appropriately to control the temperature throughout the cultivation process.

In one embodiment, the moisture level of the mixture should be suitable for the microorganism of interest. In a further embodiment, the moisture level may range from 20% to 90%, preferably, from 30 to 80%, more preferably, from 40 to 60%.

The pH of the mixture should be suitable for the microorganism of interest. Buffers, and pH regulators, such as carbonates and phosphates, may be used to stabilize pH near a preferred value. For example, the culture can be grown in a pH range from about 2 to 10 and, more specifically, at a pH range of from about 3 to 5 (by manually or automatically adjusting pH using bases, acids, and buffers; e.g., HC1, KOH, NaOH, H₃P0₄). The invention can also be practiced outside of this pH range.

Preferable results may be achieved by keeping the dissolved oxygen concentration above 10, 15, 20, or 25% of saturation during cultivation. Additionally, when metal ions are present in high concentrations, use of a chelating agent in the liquid medium may be necessary.

In preferred embodiments, the fermentation system operates continuously throughout the process of cultivation. The system can be operated for as long as necessary to produce a sufficient volume of culture, depending on the particular microbe species being produced. For example, the system can be run continuously for multiple days. In specific embodiments, the system is run continuously for 1, 2, 3, 4, or up to 5 days or more, or, if desired, until the culture has reached at least 80% sporulation.

In certain embodiments of the subject invention, a microbe growth facility produces fresh, high-density microorganisms and/or microbial growth by-products of interest on a desired scale. The microbe growth facility may be located at or near the site of application. For example, the microbe growth facility may be less than 300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from the location of use. Advantageously, this allows for the compositions to be tailored for use at a specified location. The formula and potency of microbe-based compositions can be customized for specific local conditions at the time of application, such as, for example, which soil type, plant and/or crop is being treated; what season, climate and/or time of year it is when a composition is being applied; and what mode and/or rate of application is being utilized.

The microbe growth facilities of the subject invention produce fresh, microbe-based compositions, comprising the microbes themselves, microbial metabolites, and/or other components of the medium in which the microbes are grown. If desired, the compositions can have a high density of vegetative cells or propagules, or a mixture of vegetative cells and propagules. The facility produces high-density microbe-based compositions in batch, quasi-continuous, or continuous cultivation.

The microbe growth facilities provide manufacturing versatility by their ability to tailor the microbe-based products to improve synergies with destination geographies. Advantageously, in preferred embodiments, the systems of the subject invention harness the power of naturally-occurring local microorganisms and their metabolic by-products.

Local production and delivery within, for example, 24 hours of fermentation results in pure, high cell density compositions and substantially lower shipping costs. Given the prospects for rapid advancement in the development of more effective and powerful microbial inoculants, consumers will benefit greatly from this ability to rapidly deliver microbe-based products.

Types of Culture Grown According to the Present Invention

The culture grown according to the subject invention can be, for example, any organism that is capable of being grown in culture, other than tissue culture, including bacteria, archaea, yeast, fungi, viruses or protozoa. The microorganism can be in the form of vegetative cells or propagules, e.g., spores (including, e.g., reproductive spores, endospores and/or exospores), conidia, cysts, mycelia, buds, seeds, or combinations and/or variations thereof. Preferably, the microorganism is single-celled and capable of forming spores.

These microorganisms may be natural, or genetically modified microorganisms. For example, the microorganisms may be transformed with specific genes to exhibit specific characteristics. The microorganisms may also be mutants of a desired strain. As used herein,“mutant” means a strain, genetic variant or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, missense mutation, nonsense mutation, deletion, duplication, frameshift mutation or repeat expansion) as compared to the reference microorganism. Procedures for making mutants are well known in the microbiological art. For example, UV mutagenesis and nitrosoguanidine are used extensively toward this end.

In one embodiment, the microorganism is an archaea, or eubacteria, including, but not limited to, Methanobacteria, Methanococci , Methanomicrobia, Methanopyri, Halobacteria, Halococci, Thermococcx, Thermoplasmata, Thermoproetei, Psychrobacter , Arthrobacter , Halomonas, Pseudomona , Hyphomonas, Sphingomonas, Archaeoglobi, Nanohaloarchaea, extremophilic archaea, such as thermophiles, halophiles, acidophiles, and psychrophiles, and combinations thereof.

In preferred embodiments, the microorganisms are bacteria, including Gram-positive and Gram-negative bacteria. Even more preferably, the microorganism is a spore-forming bacteria. The bacteria may be, for example, Acetonema, Actonomyces, Alkalibacillus, Ammoniphilus , Amphibacillus, Anaerobacter, Anaerospora, Aneurinibacillus, Anoxy bacillus, Bacillus (e.g., B. subtilis, B. cereus, B. licheniformis, B. firmus, B. laterosporus, B. megaterium, B. amyl ol iquefaciens and/or B. coagulans GBI-30 (BC30)), Brevibacillus, Caldanaerobacter, Caloramator, Caminicella, Cerasibacillus, Clostridium (e.g., C. bifermentans, C. butyricum , C. tetani, C. tertium, C. perfringens, C. tyrobutyricum , C. acetobutyricum , Clostridium NIPER 7, and C. beijerinckii ), Clostridiisalibacter, Cohnella, Coxiella burnetii, Dendrosporobacter, Desulfotomaculum, Desulfosporosinus, Desulfovirgula, Desulfunispora, De sulfur ispora, Filifactor, Filobacillus, Gelria, Geobacillus, Geosporobacter, Gracilibacillus, Halobacillus, Halonatronum, Heliobacterium, Heliophilum, Laceyella, Lentibacillus, Lysinibacillus, Mahella, Metabacterium, Moorella, myxobacteria (e.g. Myxococcus xanthus), Natroniella, Oceanobacillus, Orenia, Omithinibacillus, Oxalophagus, Oxobacter, Paenibacillus, Paraliobacillus, Pelospora, Pelotomaculum, Piscibacillus, Planifilum, Pontibacillus, Propionispora, Salinibacillus, Salsuginibacillus, Seinonella, Shimazuella, Sporacetigenium, Sporoanaerobacter, Sporobacter, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa, Sporosarcina, Sporotalea, Sporotomaculum, Syntrophomonas, Syntrophospora, Tenuibacillus, Tepidibacter, Terribacillus, Thalassobacillus, Thermoacetogenium, Thermoactinomyces, Thermoalkalibacillus, Thermoanaerobacter, Thermoanaeromonas, Thermobacillus, Thermoflavimicrobium, Thermovenabulum, Tuberibacillus, Virgibacillus and Vulcanobacillus.

In one embodiment, the microbe is a strain of Bacillus, e.g., B. subtilis, B. licheniformis, B. firmus, B. laterosporus, B. megaterium, B. amyloliquifaciens and/or Bacillus coagulans GBI-30 (BC30).

In one embodiment, microorganism is a strain of B. subtilis, such as, for example, B. subtilis var locuses B1 or B2, which are effective producers of, for example, surfactin and other biosurfactants, as well as biopolymers. This specification incorporates by reference International Publication No. WO 2017/044953 A1 to the extent it is consistent with the teachings disclosed herein.

A culture of the B. subtilis B1 microbe has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 USA. The deposit has been assigned accession number ATCC No. PTA-123459 by the depository and was deposited on August 30, 2016.

In certain embodiments, the present invention utilizes Bacillus subtilis strains with enhanced biosurfactant production compared to wild type Bacillus subtilis as well as compared to other microbes used in oil recovery. Such Bacillus subtilis have been termed members of the B series, including, but not limited to, Bl, B2 and B3.

In certain embodiments, the Bacillus subtilis strains are salt tolerant. Salt tolerance can be with respect to any one or more of a variety of salts. For example, the salt can be a monovalent salt such as a sodium or potassium salt, e.g., NaCl or KC1, or a divalent salt such as a magnesium or calcium salt, e.g., MgCl₂ or CaCl₂, or a trivalent salt. Given geographic sites to be treated, zinc, bromium, iron, or lithium salts are present in the composition or site. In preferred embodiments, the bacteria described herein are tolerant to NaCl as well as others of the aforementioned salts and are, therefore, widely useful for oil recovery.

In preferred embodiments, such strains are characterized by enhanced biosurfactant production compared to wild type Bacillus subtilis strains. In certain embodiments, the Bacillus subtilis strains have increased biopolymer solvent and/or enzyme production.

In certain embodiments, the microbe used according to the subject invention is Bacillus licheniformis . B. licheniformis is a Gram-positive, mesophilic bacterium, capable of anaerobic growth. It can survive in harsh environments, and at temperatures ranging from 10 to 55 °C or higher, with optimal growth temperature around 50 °C.

Bacillus licheniformis is an effective producer of biosurfactants, as well as biopolymers, including, for example, levan.

In certain embodiments, the microbe is the probiotic Bacillus coagulans GBI-30 (BC30). BC30 has been shown to promote digestive health, aide in reducing inflammation, and regulate imbalances in lipid metabolism and the immune system. BC30 is capable of surviving the acidity of the stomach, thus allowing it to reach the intestines. It contains a natural protective layer of proteins, which allows it to not only survive the harsh environment of the stomach, but also allows it to survive most manufacturing processes. Moreover, BC30 may also out-compete other harmful bacteria that cause infections or may have other deleterious effects. BC30 may delay the onset of symptoms and promote quicker recovery from infection and/or colitis caused by Clostridium difficile. It may also be helpful in replenishing beneficial bacteria in the intestines for individuals who have been prescribed antibiotics.

In one embodiment, a single type of microbe is grown in the fermentation reactor (e.g., a mixing apparatus as described herein). In alternative embodiments, multiple microbes, which can be grown together without deleterious effects on growth or the resulting product, can be grown together in a single vessel. There may be, for example, 2 to 3 or more different microbes grown at the same time.

Microbe-Based Products and Uses Thereof

The subject invention further provides microbe-based products, as well as uses for these products to achieve beneficial results in many settings including, for example, improved bioremediation and mining; waste disposal and treatment; enhancing livestock and other animal health; and promoting plant health and productivity by applying one or more of the microbe-based products.

The microbe-based products of the subject invention include products comprising the microbes and/or microbial growth by-products and optionally, the growth medium and/or additional ingredients such as, for example, water, carriers, adjuvants, nutrients, viscosity modifiers, and other active agents.

One microbe-based product of the subject invention is the reference culture comprising sporulated microorganisms, nutrient medium and spore stabilizer. One microbe-based product is an inoculum comprising an aliquot of reference culture.

Yet another microbe-based product is obtained by the cultivation of the inoculum of reference culture, and can comprise the microorganism, residual nutrient medium, and/or any growth by products of the microorganism. The product of cultivation may be used directly without extraction or purification. If desired, extraction and purification can be easily achieved using standard extraction methods or techniques known to those skilled in the art.

The microorganisms may be in an active or inactive form. Advantageously, in accordance with the subject invention, the microbe-based product may comprise broth in which the microbes were grown. The product may be, for example, at least, by weight, 1%, 5%, 10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product, by weight, may be, for example, anywhere from 0% to 100% inclusive of all percentages there-between.

The subject invention further provides materials and methods for the production of biomass ( e.g ., viable cellular material), extracellular metabolites and solvents (e.g., both small and large molecules), and/or intracellular components (e.g., enzymes and other proteins). The microbes and microbial growth by-products of the subject invention can also be used for the transformation of a substrate, such as an ore, wherein the transformed substrate is the product.

In one embodiment, the subject invention provides a method of improving plant health and/or increasing crop yield by scaling the microbe-based product disclosed herein, for example in an on-site fermentation system, and applying the scaled product to soil, seed, or plant parts. In another embodiment, the subject invention provides a method of increasing crop or plant yield comprising multiple applications of the scaled product.

In one embodiment, the subject invention provides methods of producing a surfactant, solvent, enzyme, and/or other useful metabolite, by cultivating a microbe strain of the subject invention under conditions appropriate for growth and such metabolite production; and, optionally, at least to some extent, the metabolite. The microorganisms can grow in situ and produce the metabolites onsite. Consequently, a high concentration of metabolites and biosurfactant-producing microorganisms at a treatment site (e.g., an oil well) can be achieved efficiently and continuously. In one embodiment, the composition is suitable for agriculture. For example, the composition can be scaled and used to treat soil, plants, and seeds. The composition may also be used as a pesticide.

In one embodiment, the subject invention further provides customizations to the materials and methods according to the local needs. For example, the method for cultivation of microorganisms may be used to grow those microorganisms located in the local soil or at a specific oil well or site of pollution. In specific embodiments, local soils may be used as the solid substrates in the cultivation method for providing a native growth environment. Advantageously, these microorganisms can be beneficial and more adaptable to local needs.

Claims

CLAIMS WE CLAIM:

1. A method of producing a reference culture comprising the steps of:

cultivating a microorganism in a first cultivation reactor having nutrient medium therein;

allowing the microorganism to sporulate; and

adding a spore stabilizer substance to the first reactor to stabilize the sporulated microorganism,

wherein the reference culture comprises the sporulated microorganism, nutrient medium and spore stabilizer substance.

2. The method of claim 1 , wherein the microorganism is a spore-forming strain of Bacillus.

3. The method of claim 2, wherein the microorganism is selected from B. subtilis, B. licheniformis, B.flrmus, B. laterosporus, B. megaterium, B. amyloliquefaciens and Bacillus coagulans GBI-30 (BC30).

4. The method of claim 1, wherein the spore stabilizer substance is 0.1 to 0.5% v/v phenol.

5. The method of claim 1 , further comprising the step of preserving the reference culture.

6. The method of claim 5, wherein preserving the reference culture comprises keeping the reference culture in the first cultivation reactor at a temperature ranging from 0°C to 25°C.

7. The method of claim 5, wherein preserving the reference culture comprises transferring portions of the reference culture into containers and keeping the containers at a temperature ranging from 0°C to 25°C.

8. The method of claim 5, wherein the reference culture is capable of being preserved for 1 month or longer.

9. A stabilized reference culture comprising a microorganism, a nutrient medium and phenol.

10. The stabilized reference culture of claim 9, produced using the methods of claim 1-8.

11. The stabilized reference culture of claim 9, used to inoculate a cultivation reactor for producing a microbe-based composition.

12. A method of producing a microbe-based composition, the method comprising obtaining, as an inoculum, an aliquot of the reference culture of claim 9, placing the inoculum into a fermentation reactor having nutrient medium therein, and cultivating the inoculum until a desired cell concentration is reached.

13. The method of claim 12, wherein the nutrient medium comprises a germination enhancer.

14. The method of claim 1 , wherein the germination enhancer is selected from L-alanine and manganese.

15. The method of claim 12, wherein cultivating the inoculum is carried out at a temperature of between lO°C and 50°C.

16. The method of claim 12, wherein the aliquot has a volume of 0.1 mΐ to 10 liters.

17. A composition comprising microorganisms cultivated by the method of claim 12 and/or at least one microbial growth by-product of said microorganisms.