NOVEL CULTIVATION SYSTEM FOR THE EFFICIENT PRODUCTION OF MICROORGANISMS

Abstract

The invention generally relates to the cultivation and growth of bacterial, fungal and yeast cells on pilot plant and industrial scales and, more particularly, to the cultivation and growth of microbial cells in/on hydrophilic particles containing pre-seeded nutrient media within a matrix of hydrophobic sand.

Claims

1. A method for cultivating microorganisms, wherein said method comprises the steps of: a) mixing a hydrophobic material and hydrophilic particles to form a matrix; b) contacting said matrix, said hydrophobic material, and/or said hydrophilic particles, with a medium inoculated with microorganisms, thereby creating a growth matrix; and c) growing the microorganisms within said growth matrix.

2. The method according to claim 1, wherein the microorganisms are bacteria.

3-4. (canceled)

5. The method according to claim 1, wherein the microorganisms are fungi.

6. (canceled)

7. The method according to claim 1, wherein the hydrophobic material and hydrophilic particles are matrix-forming materials and are mixed with the medium inoculated with the microorganism at a ratio ranging from 20:1 to 5:1 of matrix-forming materials to medium.

8-9. (canceled)

10. The method, according to claim 1, wherein the hydrophilic particles have a water contact angle of 90 or less.

11. The method, according to claim 1, wherein the hydrophilic particles have a porosity of at least about 5%.

12. The method, according to claim 1, wherein the hydrophilic particles are selected from perlite, vermiculite, metal-containing ores, and diatomaceous earth.

13. The method according to claim 1, wherein the hydrophobic material is hydrophobic sand.

14. The method, according to claim 13, wherein the hydrophobic sand is coated with an organosilicon compound.

15-18. (canceled)

19. A composition comprising microorganisms, hydrophobic sand and hydrophilic particles wherein the hydrophilic particles are approximately 0.0001 to 10 mm in diameter and have a water contact angle of 90 or less, and wherein the microorganisms are present at a concentration of at least 10.sup.4 CFU/ml.

20. The composition, according to claim 19, wherein the hydrophilic particles have a porosity of at least about 5%.

21. The composition, according to claim 19, wherein the hydrophobic sand is coated with an organosilicon compound.

22-25. (canceled)

26. The composition, according to claim 19, wherein the microorganisms are bacteria.

27-28. (canceled)

29. The composition, according to claim 19, wherein the microorganisms are fungi.

30. The composition, according to claim 29, wherein the fungi are Mycorrhizal or Starmerella.

31. A method for enhancing the amount of oil recoverable from an oil-containing formation, wherein said method comprises applying a composition of claim 19 to the oil-containing formation.

32. (canceled)

33. A method for cleaning an oil well rod, tubing and/or casing, wherein said method comprises applying to the oil well rod, tubing and casing structures a composition of claim 19.

34. A method for improving plant growth, yield, and/or health, wherein said method comprises applying to the plant or its environment a composition of claim 19.

35-39. (canceled)

40. The method, according to claim 34, wherein the composition has activity against an insect pest of agriculture, turf, ornamentals, forestry and/or other plant based production.

41. The method, according to claim 34, wherein the composition has activity against weeds.

42. A method for controlling a pest of a structure wherein said method comprises applying to the structure, or the vicinity of the structure, or directly to the pest, a composition of claim 19.

43. A method for controlling a pest of animals wherein said method comprises contacting the pest with a composition of claim 19.

44-45. (canceled)

46. A method for bioleaching an ore wherein said method comprises administering to the ore a composition of claim 19.

47-48. (canceled)

Description

EXAMPLE 1

Bacterial Cultivation with Vermiculite as a Hydrophilic Substrate

[0126] Vermiculite, a hydrated magnesium aluminum silicate, is a cheap and commonly used product in gardening that is very porous.

[0127] The cultivation of Bacillus spp was carried out in a vessel where a small amount of liquid broth inoculum was introduced to the mixture of vermiculite and hydrophobic sand at a ratio of 1:12. This growth matrix was then mixed to homogeneity. The containing vessel was then incubated and received daily aeration treatments with ambient air.

[0128] Alternatively, a small amount of liquid broth inoculum (e.g., 10.sup.5 CFU/mL or 10.sup.4 CFU/mL) is mixed with the vermiculite in a 1/5 volumetric ratio. This mixture is blended well, leaving individual particles coated with inoculum. This mixture is further combined with a hydrophobic sand in a ratio ranging from 5:1 to 12:1 sand to inoculum, by volume, in an appropriate growth vessel containing aeration ports.

[0129] This can be further homogenized by agitation or mixing. The vessel is then incubated at appropriate temperatures and aerated at least three times per day.

[0130] To quantify cell density, a daily sample was taken and enumerated following serial dilution plating technique. Briefly, serial dilution of the sample is obtained. When fixed volumes of this dilution series are spread onto a solid growth medium and incubated, different numbers of colonies will be obtained. By noting the number of colonies, the volume of inoculant added, and the mass or volume of sample diluted, the number of microorganisms in the original sample can be calculated.

[0131] Under the conditions given in this example, in two days of cultivation, the cell concentration was grown from 10.sup.4 to at least 10.sup.9 CFU/mL.

EXAMPLE 2

Bacterial Cultivation with Perlite as a Hydrophilic Substrate

[0132] Perlite is a highly porous volcanic glass derived from obsidian. It is often used in insulation and gardening applications.

[0133] Following the same procedure as previously described for vermiculite, an optimal method was developed for the cultivation of Bacillus spp. utilizing perlite as a hydrophilic substrate. This involves adding liquid inoculum at a ratio of 1:12 to a perlite and hydrophobic sand mixture, which is homogenized and incubated.

[0134] Alternatively, a small amount of liquid broth inoculum (e.g., 10.sup.5 CFU/mL or 10.sup.4 CFU/mL) is mixed with the vermiculite in a 1/5 volumetric ratio. This mixture is blended well, leaving small individual saturated particles. This mixture is further combined with the hydrophobic sand matrix at a ratio from 5:1 to 12:1 sand to inoculum, by volume, in an appropriate growth vessel containing aeration ports.

[0135] This can be further homogenized by agitation or mixing. The vessel is then incubated at appropriate temperatures and aerated at least three times per day.

[0136] To quantify cell density, a daily sample was taken and enumerated following serial dilution plating technique.

[0137] Enumeration sampling showed that this method is also capable of reaching (from 10.sup.4) viable counts of at least 10.sup.9 CFU/mL within 2-3 days of inoculation.

EXAMPLE 3

Cultivation of Mycorrhiza Fungi with Diatomaceous Earth as a Hydrophilic Substrate

[0138] A method for the cultivation of Mycorrhiza utilizing hydrophobic sand and diatomaceous earth was also developed.

[0139] In this instance, a culture containing 10.sup.5 CFU/mL of the fungus is combined in a 1/5 ratio, by volume, to diatomaceous earth and blended to a fine particulate consistency. This is then further combined with the hydrophobic sand at a ratio from 5:1 to 12:1 sand to inoculum, by volume, preferably, 6:1 sand to inoculum ratio.

[0140] This mixture was allowed to incubate under optimal fungal growth conditions in a growth vessel, typically an appropriately sized glass bottle with aeration receptacles, and further shaken to homogeneity.

[0141] The sample was enumerated daily, yielding at least 10,000 propagules/gram (with initial concentration of 150 propagules/gram) within 2 weeks of inoculation.

EXAMPLE 4

Production of Microorganisms for Agriculture

[0142] The methods of the subject invention can be used to generate large amounts of concentrated cultures to be used for agricultural applications. Many examples of microbial application for agricultural purposes exist, including using Bacillus, Pseudomonas, and Mycorrhizae strains.

[0143] In certain embodiments, the method of the subject invention facilitates the production of large quantities of bacteria at the site in a close distance to application. The microbes can be grown on site and utilized still within the growth matrix. The microorganisms will integrate into the soil and microbes will be present in sufficient quantities to improve plant health and growth.

[0144] In one embodiment, the subject invention provides microbe-based compositions, as well as methods of using the compositions for promoting plant health, soil microbial diversity, plant nutrition, soil nutritive capacity, optimizing soil moisture status, soil aeration, soil water holding capacity and reducing the susceptibility of plants, to pests, diseases and weeds. This is achieved by improving a plant's natural defenses, the nutritive content, the microbial and toxilogical health of soils as well as by directly impacting plant pests, diseases or weeds. This plant-promotion effect occurs as a result of applying one or more of the microbe-based products of the subject invention to the plant and/or its environment.

[0145] In one embodiment, the compositions can be used to promote plant growth, yield and/or health. The composition may have activity against, for example, fungi and/or bacterial plant pathogens. The composition may also have activity against weeds. The composition may have activity against an insect pest of agriculture, turf, ornamentals, forestry and/or plant based production. The composition can be added to horticultural soil mixes; added at the time of planting of seed or transplants; or broadcast by hand or machine to agricultural fields, ponds, forests or any environment where it is desired to impact crops, animals and their pests.

EXAMPLE 5

Bioleaching

[0146] The present invention can be used in the process of bio-leaching. In this case, the process involves the cultivation of microorganisms capable of accumulating metals inside the cells. For example, Cupriavidus matallidurans can solubilize gold in ore to a soluble ionic form and convert into nanoparticles inside a cell.

[0147] Gold-containing ore can be used as micro-particles wetted with an appropriate nutrient medium for growing the bacterium in the matrix of hydrophobic sand.

[0148] Because this method does not require complicated equipment and high energy consumption, the installation for cultivation can be built at a site of ore.

EXAMPLE 6

Bioremediation

[0149] The present invention can be used to grow substantial quantities of bacteria and/or fungi that can be used to bio-remediate polluted soils and water.

[0150] Many microorganisms can be used for decontamination through bioremediation, including, for example, Pseudomonas, Arthrobacter and Bacillus strains. The microorganisms can be grown on site and released being still encapsulated in the hydrophobic matrix. The decontaminating microorganisms will be present in high numbers when introduced into the contaminated soil or water.

EXAMPLE 7

Oil Production

[0151] The subject invention provides microbe-based products, as well as their uses, in improved oil production. In certain embodiments, the subject invention provides materials and methods for improving oil production by treating drilling sites, including the wells and associated piping, with microorganisms and/or their by-products in order to enhance recovery of oil.

[0152] In some embodiments, the microbes can be salt-tolerant and/or surfactant over-producing microbes and by-products thereof. These by-products can include, for example, metabolites, polymers, biosurfactants, enzymes, carbon dioxide, organic acids, and solvents.

[0153] In preferred embodiments, such strains are characterized by enhanced biosurfactant production compared to wild type strains.

EXAMPLE 8

Pests of Structures

[0154] In one embodiment, the compositions of the subject invention have activity against pests of structures. These pests can be, for example, termites, ants or roaches. To control such pests, the composition can be applied, directly to the pests, or to the structure or the vicinity of the structure such that the pest will come into contact with the composition.

EXAMPLE

Pests of Animals

[0155] In another embodiment, the compositions of the subject invention can be used to control pests of animals, including humans. These pests can be, for example, mosquitoes, flies, nematodes, ticks, and fleas. To control such pests, the composition can be applied, directly to the pests, or to their environment such that the pest will come into contact with the composition. The microbes can be, for example, Bacillus thuriengensis. Control can include killing as well as reducing eating reproduction or other activity.

[0156] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0157] The description herein of any aspect or embodiment of the invention using terms such as comprising, having, including, or containing with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that consists of, consists essentially of, or substantially comprises that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0158] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.