Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
MICROBE COMBINATIONS FOR BIOREMEDIATION AND METHODS OF USING THE SAME
Document Type and Number:
WIPO Patent Application WO/2019/118982
Kind Code:
A1
Abstract:
Novel combinations of Bacillus strains are provided, including combinations suitable for bioremediation of contaminants. The disclosure also relates to compositions comprising the novel combinations of Bacillus strains and methods of using the same to bioremediate contaminated sites.

Inventors:
PENET, Christopher (6136 Wolverhampton Dr, Raleigh, North Carolina, 27693, US)
SPEARS, Jessica (6285 Heathbrook Dr, Eden Prairie, Minnesota, 55346, US)
LAMB, Steve (801 Western Air Dr, Jefferson City, Missouri, 65109, US)
LAWSON III, Joseph Stebbins (1112 N. Rotary Drive, High Point, North Carolina, 27262, US)
BALLSIEPER, Jeffrey Allen (120 Staffordshire Court, Winston-Salem, North Carolina, 27104, US)
Application Number:
US2018/066071
Publication Date:
June 20, 2019
Filing Date:
December 17, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
BIO-CAT MICROBIALS LLC (689 Canterbury Road, Shakopee, Minnesota, 55379, US)
International Classes:
C02F3/34; B09C1/00; B09C1/10; C02F1/00; C02F3/00
Attorney, Agent or Firm:
SPIEGLER, Alexander et al. (1717 K Street NWWashington, District of Columbia, 20006, US)
Download PDF:
Claims:
CLAIMS

1. A composition comprising a blend of cells or endospores from multiple Bacillus strains, wherein the strains comprise: B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis MO 17, 1E9 (PTA-121389); B. licheniformis 07SI, 1E9; B. amyloliquefaciens 2E9 (PTA-121388); B. pumilus 1875, 1E9 (NRRL-1875); B. pumilus 07SI, 1E9; B. laterosporus CM33 2E9 (PTA-3592); B. laterosporus CM3 (PTA-3593); B. subtilis 10SI, 1E9; B. subtilis 1109, 1E9 (ATCC-9524); and . mojavensis 1E8.

2. The composition of claim 1, wherein the composition is capable of reducing an amount or concentration of at least one contaminant when placed in contact with the at least one contaminant.

3. The composition of claims 1 or 2, wherein the composition is capable of reducing an amount or concentration of at least one contaminant when applied to a contaminated area.

4. The composition of claim 3, wherein the contaminated area comprises sediment, sand, gravel, soil, groundwater, aquifer material, or landfill.

5. The composition of claims 3 or 4, wherein the contaminated area comprises: soil or groundwater.

6. The composition of any one of claims 2-5, wherein the at least one contaminant comprises one or more of the following: crude oil, gasoline, diesel fuel, heating oil, and/or mineral oil.

7. The composition of any one of claims 2-6, wherein the at least one contaminant comprises at least one organic compound that has been distilled and/or derived from petroleum.

8. The composition of any one of claims 2-7, wherein the at least one contaminant comprises one or more of the following:

(a) an aliphatic or aromatic hydrocarbon;

(b) a polycyclic aromatic hydrocarbon (PAH);

(c) a substituted hydrocarbon; (d) a halogenated or oxygenated hydrocarbon;

(e) an organic compound;

(f) a semi-volatile organic compound (SVOC);

(g) a volatile organic compound (VOC); and/or

(h) benzene, toluene, ethylbenzene or xylene.

9. The composition of any one of claims 2-8, wherein the at least one contaminant comprises a semi-volatile organic compound (SVOC) or a polycyclic aromatic hydrocarbon (PAH).

10. The composition of claim 9, wherein the SVOC or PAH comprises one or more of the following: acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, 4-bromophenylphenyl ether, butylbenzylphthalate, 4-chloro-3-methylphenol, bis(2-chloroethoxy)methane, bis(2-chloroethyl) ether, 2-chloronaphthalene, 2-chlorophenol, 4-chlorophenylphenyl ether, chrysene,

dibenz(a,h)anthracene, 3,3'-dichlorobenzidine, 2,4-dichlorophenol, diethylphthalate, 2,4- dimethylphenol, dimethylphthalate, di-n-butylphthalate, 4,6-dinitro-2-methylphenol, 2,4- dinitrophenol, 2,4-dinitrotoluene, 2,6-dinitrotoluene, di-n-octylphthalate, bis(2- ethylhexyl)phthalate, fluoranthene, fluorene, hexachloro-l, 3-butadiene, hexachlorobenzene, hexachlorocyclopentadiene, hexachloroethane, indeno(l,2,3-cd)pyrene, isophorone, naphthalene, nitrobenzene, 2-nitrophenol, 4-nitrophenol, n-nitrosodimethylamine, n-nitroso-di-n-propylamine, n-nitrosodiphenylamine, 2,2'-oxybis(l-chloropropane), pentachlorophenol, phenanthrene, phenol, pyrene, 1, 2, 4-tri chlorobenzene or 2,4,6-trichlorophenol.

11. The composition of any one of claims 2-8, wherein the at least one contaminant comprises a volatile organic compound (VOC).

12. The composition of claim 11, wherein the VOC comprises one or more of the following petroleum hydrocarbons: benzene, bromobenzene, bromoform, bromom ethane, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, l,2-dibromoethane (EDB), dibromomethane, diisopropyl ether, ethylbenzene, hexachloro-l, 3-butadiene, isopropylbenzene (cumene), methylene chloride, methyl -tert-butyl ether, naphthalene, n-propylbenzene, styrene, toluene, l,2,4-trimethylbenzene, l,3,5-trimethylbenzene, o-xylene, m-xylene, or p-xylene.

13. The composition of claim 11, wherein the VOC comprises one or more of the following chlorinated compounds: bromochlorom ethane, bromodichlorom ethane, chlorobenzene, chloroethane, chloroform, chlorom ethane, l,2-dibromo-3-chloropropane,

dibromochloromethane, 1 ,2-di chlorobenzene, 1, 3 -di chlorobenzene, 1 ,4-di chlorobenzene, dichlorodifluoromethane, l,2-dichloropropane, l,3-dichloropropane, 2,2-dichloropropane, 1,1- dichloropropene, cis-l,3-dichloropropene, trans-l,3-dichloropropene, 2-chlorotoluene, 4- chlorotoluene, carbon tetrachloride, 1,1 -di chloroethane, 1 ,2-di chloroethane, l,l-dichloroethene, cis-l,2-dichloroethene, trans-l,2-dichloroethene, tetrachloroethene, 1,2, 3-trichlorobenzene,

1, 2, 4-tri chlorobenzene, trichlorofluoromethane, l,2,3-trichloropropane, 1,1,1 -tri chloroethane,

1 , 1 ,2-tri chloroethane, l,l,l,2-tetrachloroethane, l,l,2,2-tetrachloroethane, trichloroethene or vinyl chloride.

14. The composition of any one of claims 2-13, wherein each of the strains in the blend is present at a concentration of:

(a) 1 x 103 to 1 x 1012 colony forming units (CFU)/mL;

(b) l x lO3 to l x lO6 CFU/mL;

(c) l x lO6 to l x lO12 CFU/mL; or

(d) l x lO9 to l x lO12 CFU/mL.

15. The composition of any one of claims 2-14, wherein the average concentration of all strains in the blend is:

(a) 1 x 103 to l x lO12 colony forming units (CFU)/mL;

(b) l x lO3 to l x lO6 CFU/mL;

(c) l x lO6 to l x lO12 CFU/mL; or

(b) l x lO9 to l x lO12 CFU/mL.

16. The composition of any one of claims 2-15, wherein the average concentration of all strains in the blend is at least: (a) 1 x 103 colony forming units (CFU)/mL;

(b) l x lO6 CFU/mL;

(c) U 109 CFU/mL; or

(d) l x lO12 CFU/mL.

17. The composition of any one of claims 2-16, wherein the at least one contaminant comprises one or more of the following:

(a) total benzene, toluene, ethylbenzene, xylenes (total“BTEX”);

(b) total xylenes;

(c) total petroleum hydrocarbons as diesel (TPH-D);

(d) total petroleum hydrocarbons as gasoline (TPH-G); and/or

(e) total petroleum hydrocarbons as oil and grease (TPH-O&G).

18. The composition of any one of claims 1-17, wherein the composition comprises only endospores.

19. The composition of any one of claims 1-18, wherein the composition is spray-dried.

20. The composition of any one of claims 2-19, wherein the composition is formulated as a

(a) slurry; and/or

(b) liquid suspension.

21. The composition of any one of claims 2-19, wherein the composition is formulated as a

(a) spray-dried powder; and/or

(b) lyophilisate.

22. The composition of any one of claims 1-21, further comprising: approximately 1-2 wt% of a sugar.

23. The composition of any one of claims 1-22, further comprising at least one surfactant or solubilizer.

24. A method for bioremediating a contaminated area, the method comprising:

(i) treating the contaminated area by applying a composition comprising a blend of cells or endospores from one or more Bacillus strains to the contaminated area; and

(ii) reducing an amount or concentration of at least one contaminant in the area;

wherein the one or more strains comprise: B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis M017, 1E9 (PTA-121389); B. licheniformis 07SI, 1E9; B. amyloliquefaciens 2E9 (PTA-121388); B. pumilus 1875, 1E9 (NRRL-1875); B. pumilus 07SI, 1E9; B. laterosporus CM33 2E9 (PTA-3592); B. laterosporus CM3 (PTA-3593); B. subtilis 10SI, 1E9; B. subtilis 1109, 1E9 (ATCC-9524); and/or B. mojavensis 1E8.

25. The method of claim 24, wherein the contaminated area comprises sediment, sand, gravel, soil, groundwater, surface water, aquifer material, or landfill.

26. The method of claims 24 or 25, wherein the contaminated area comprises soil or

groundwater.

27. The method of any one of claims 24-26, wherein the at least one contaminant comprises one or more of the following: crude oil, gasoline, diesel fuel, kerosene, hydraulic oil, motor oil, heating oil, or mineral oil.

28. The method of any one of claims 24-27, wherein the at least one contaminant comprises at least one organic compound that has been distilled and/or derived from petroleum.

29. The method of any one of claims 24-28, wherein the at least one contaminant comprises one or more of the following: crude oil, gasoline, diesel fuel, kerosene, hydraulic oil, motor oil, heating oil, or mineral oil.

30. The method of any one of claims 24-29, wherein the at least one contaminant comprises one or more of the following:

(a) an aliphatic or aromatic hydrocarbon; (b) a polycyclic aromatic hydrocarbon (PAH);

(c) a substituted hydrocarbon;

(d) a halogenated or oxygenated hydrocarbon;

(e) an organic compound;

(f) a semi-volatile organic compound (SVOC);

(g) a volatile organic compound (VOC); and/or

(h) benzene, toluene, ethylbenzene or xylene.

31. The method of any one of claims 24-30, wherein the at least one contaminant comprises a semi-volatile organic compound (SVOC) or a polycyclic aromatic hydrocarbon (PAH).

32. The method of claim 31, wherein the SVOC or PAH comprises one or more of the following: acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene,

benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, 4-bromophenylphenyl ether, butylbenzylphthalate, 4-chloro-3-methylphenol, bis(2-chloroethoxy)methane, bis(2-chloroethyl) ether, 2-chloronaphthalene, 2-chlorophenol, 4-chlorophenylphenyl ether, chrysene,

dibenz(a,h)anthracene, 3,3'-dichlorobenzidine, 2,4-dichlorophenol, diethylphthalate, 2,4- dimethylphenol, dimethylphthalate, di-n-butylphthalate, 4,6-dinitro-2-methylphenol, 2,4- dinitrophenol, 2,4-dinitrotoluene, 2,6-dinitrotoluene, di-n-octylphthalate, bis(2- ethylhexyl)phthalate, fluoranthene, fluorene, hexachloro-l, 3-butadiene, hexachlorobenzene, hexachlorocyclopentadiene, hexachloroethane, indeno(l,2,3-cd)pyrene, isophorone, naphthalene, nitrobenzene, 2-nitrophenol, 4-nitrophenol, n-nitrosodimethylamine, n-nitroso-di-n-propylamine, n-nitrosodiphenylamine, 2,2'-oxybis(l-chloropropane), pentachlorophenol, phenanthrene, phenol, pyrene, 1, 2, 4-tri chlorobenzene or 2,4,6-trichlorophenol.

33. The method of any one of claims 24-30, wherein the at least one contaminant comprises a volatile organic compound (VOC).

34. The method of claim 33, wherein the VOC comprises one or more of the following petroleum hydrocarbons: benzene, bromobenzene, bromoform, bromom ethane, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, l,2-dibromoethane (EDB), dibromomethane, diisopropyl ether, ethylbenzene, hexachloro-l, 3-butadiene, isopropylbenzene (cumene), methylene chloride, methyl -tert-butyl ether, naphthalene, n-propylbenzene, styrene, toluene, l,2,4-trimethylbenzene, l,3,5-trimethylbenzene, o-xylene, m-xylene, or p-xylene.

35. The method of claim 33, wherein the VOC comprises one or more of the following chlorinated compounds: bromochlorom ethane, bromodichlorom ethane, chlorobenzene, chloroethane, chloroform, chlorom ethane, l,2-dibromo-3-chloropropane,

dibromochloromethane, 1 ,2-di chlorobenzene, 1, 3 -di chlorobenzene, 1 ,4-di chlorobenzene, dichlorodifluoromethane, l,2-dichloropropane, l,3-dichloropropane, 2,2-dichloropropane, 1,1- dichloropropene, cis-l,3-dichloropropene, trans-l,3-dichloropropene, 2-chlorotoluene, 4- chlorotoluene, carbon tetrachloride, 1,1 -di chloroethane, 1 ,2-di chloroethane, l,l-dichloroethene, cis-l,2-dichloroethene, trans-l,2-dichloroethene, tetrachloroethene, 1,2, 3-trichlorobenzene,

1, 2, 4-tri chlorobenzene, trichlorofluoromethane, l,2,3-trichloropropane, 1,1,1 -tri chloroethane,

1 , 1 ,2-tri chloroethane, l,l,l,2-tetrachloroethane, l,l,2,2-tetrachloroethane, trichloroethene or vinyl chloride.

36. The method of any one of claims 24-35, wherein the at least one contaminant in the soil or groundwater comprises:

(a) total benzene, toluene, ethylbenzene, xylenes (total“BTEX”);

(b) total xylenes;

(c) total petroleum hydrocarbons as diesel (TPH-D);

(d) total petroleum hydrocarbons as diesel (TPH-D);

(e) total petroleum hydrocarbons as gasoline (TPH-G); and/or

(f) total petroleum hydrocarbons as oil and grease (TPH-O&G).

37. The method of any one of claims 24-36, wherein the composition is applied to the contaminated area as a:

(a) slurry; and/or

(b) suspended in a liquid formulation.

38. The method of any one of claims 24-36, wherein the composition is applied to the contaminated area as a:

(a) spray-dried formulation; and/or

(b) lyophilisate.

39. The method of any one of claims 24-38, wherein the amount or concentration of the at least one contaminant is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 99% after treatment compared to an untreated sample of the contaminated area after 30, 60, 90 or 120 days.

40. The method of any one of claims 24-39, wherein the concentration of cells or endospores of each of the Bacillus strains in the composition applied to the contaminated area is:

(a) at least U103, IcIO4, 1c105, IcIO6, IcIO7, IcIO8, IcIO9, IcIO10, lxlO11, or lxlO12 colony forming units (CFU)/mL;

(b) from lxlO3 CFU/ml to lxlO12;

(c) from lxlO6 CFU/ml to 1 x 109;

(d) from lxlO9 CFU/ml to 1 1012 ; and/or

(e) from UlO3 CFU/ml to lxlO6.

41. The method of any one of claims 24-39, wherein the average concentration of cells or endospores of all of the Bacillus strains in the composition applied to the contaminated area is:

(a) at least UlO3, UlO4, UlO5, UlO6, U107, U108, UlO9, UlO10, lxlO11, or UlO12 colony forming units (CFU)/mL;

(b) from lxlO3 CFU/ml to UlO12;

(c) from lxlO6 CFU/ml to 1 x 109;

(d) from lxlO9 CFU/ml to 1 1012 ; and/or

(e) from UlO3 CFU/ml to UlO6.

42. The method of any one of claims 24-41, further comprising: repeating steps (i) and (ii) at least once.

43. The method of any one of claims 24-42, wherein the amount or concentration of the at least one contaminant in the contaminated area is reduced by at least 95%, 96%, 97%, 98%, or 99% compared to an untreated sample of the area after 30 days or 60 days.

44. The method of any one of claims 24-43, wherein the at least one contaminant is benzene, ethylbenzene, xylenes, and/or toluene and the contaminated area comprises soil or groundwater; and the concentration of benzene, ethylbenzene, xylenes, and/or or toluene in the soil or groundwater is reduced by at least 90%, 95% or 99% compared to an untreated sample of the soil or groundwater after 30, 60 or 90 days.

45. The method of any one of claims 24-44, wherein the composition does not contain any enzymes, surfactants, or solublizers except for those produced by the Bacillus strains.

46. A method for bioremediating contaminated groundwater, comprising:

placing a pipe within proximity to a contaminant plume that comprises at least one contaminant; and

applying a suspension comprising a blended mixture of Bacillus cells or endospores to the contaminant plume using the pipe;

wherein the blended mixture of Bacillus cells or endospores is applied in an amount sufficient to reduce the concentration of the contaminant in the groundwater.

47. The method of claim 46, wherein the suspension is applied to the contaminant plume using the pipe under pressure.

48. The method of claims 46 or 47, wherein the contaminant comprises one or more of the following:

(a) a semi-volatile organic compound (SVOC) or a polycyclic aromatic hydrocarbon (PAH); and/or

(b) a volatile organic compound (VOC).

49. The method of claims 48, wherein the SVOC or PAH comprises one or more of the following: acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, 4-bromophenylphenyl ether, butylbenzylphthalate, 4-chloro-3-methylphenol, bis(2-chloroethoxy)methane, bis(2-chloroethyl) ether, 2-chloronaphthalene, 2-chlorophenol, 4-chlorophenylphenyl ether, chrysene,

dibenz(a,h)anthracene, 3,3'-dichlorobenzidine, 2,4-dichlorophenol, diethylphthalate, 2,4- dimethylphenol, dimethylphthalate, di-n-butylphthalate, 4,6-dinitro-2-methylphenol, 2,4- dinitrophenol, 2,4-dinitrotoluene, 2,6-dinitrotoluene, di-n-octylphthalate, bis(2- ethylhexyl)phthalate, fluoranthene, fluorene, hexachloro-l, 3-butadiene, hexachlorobenzene, hexachlorocyclopentadiene, hexachloroethane, indeno(l,2,3-cd)pyrene, isophorone, naphthalene, nitrobenzene, 2-nitrophenol, 4-nitrophenol, n-nitrosodimethylamine, n-nitroso-di-n-propylamine, n-nitrosodiphenylamine, 2,2'-oxybis(l-chloropropane), pentachlorophenol, phenanthrene, phenol, pyrene, 1, 2, 4-tri chlorobenzene or 2,4,6-trichlorophenol.

50. The method of claims 48, wherein the VOC comprises one or more of the following petroleum hydrocarbons: benzene, bromobenzene, bromoform, bromom ethane, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, l,2-dibromoethane (EDB), dibromomethane, diisopropyl ether, ethylbenzene, hexachloro-l, 3-butadiene, isopropylbenzene (cumene), methylene chloride, methyl -tert-butyl ether, naphthalene, n-propylbenzene, styrene, toluene, l,2,4-trimethylbenzene, l,3,5-trimethylbenzene, o-xylene, m-xylene, or p-xylene

51. The method of claims 48, wherein the VOC comprises one or more of the following chlorinated compounds: bromochlorom ethane, bromodichlorom ethane, chlorobenzene, chloroethane, chloroform, chlorom ethane, l,2-dibromo-3-chloropropane,

dibromochloromethane, 1 ,2-di chlorobenzene, 1, 3 -di chlorobenzene, 1 ,4-di chlorobenzene, dichlorodifluoromethane, l,2-dichloropropane, l,3-dichloropropane, 2,2-dichloropropane, 1,1- dichloropropene, cis-l,3-dichloropropene, trans-l,3-dichloropropene, 2-chlorotoluene, 4- chlorotoluene, carbon tetrachloride, 1,1 -di chloroethane, 1 ,2-di chloroethane, l,l-dichloroethene, cis-l,2-dichloroethene, trans-l,2-dichloroethene, tetrachloroethene, 1,2, 3-trichlorobenzene,

1,2, 4-tri chlorobenzene, trichlorofluoromethane, l,2,3-trichloropropane, 1,1,1 -tri chloroethane, l,l,2-trichloroethane, l,l,l,2-tetrachloroethane, l,l,2,2-tetrachloroethane, trichloroethene or vinyl chloride.

52. The method of any one of claims 46-51, wherein the suspension applied to the contaminant plume further comprises a sugar capable of being metabolized by the blend of Bacillus cells or endospores.

53. The method of any one of claims 46-52, wherein the concentration of the contaminant in the groundwater is reduced by at least 80%, 90%, 95% or 99% after 30, 60 or 90 days.

Description:
MICROBE COMBINATIONS FOR BIOREMEDIATION AND

METHODS OF USING THE SAME

Cross-Reference to Related Patent Application

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/599,456, filed December 15, 2017, the disclosure of which is herein incorporated by reference in its entirety.

Technical Field

[0002] Novel combinations of Bacillus strains are provided, including combinations suitable for bioremediation of contaminants ( e.g ., petroleum-related and other organic compounds). Compositions comprising these novel combinations and methods of using the same to bioremediate contaminated sites are also provided.

Background

[0003] The remediation of contaminated soil and groundwater has become increasingly important in recent years due to the enactment of stringent environmental laws and increased public awareness of health concerns associated with exposure to contaminated sites. The widespread use of petroleum and its derivatives (e.g., gasoline, diesel fuel, heating oil, kerosene, hydraulic oil, motor oil (used and new), and mineral oil) is a particular cause for concern due to the potential for leakage from underground storage tanks and spillage or improper disposal of these chemicals. This may result in contamination of soil and/or groundwater located at (or near) the site. Organic compounds obtained from petroleum (e.g., aliphatic and aromatic hydrocarbons, as well as other substituted derivatives) are also commonly used industrial solvents and chemical intermediates in a variety of chemical processes. [0004] Unintentional spillage of petroleum-based distillates/derivatives, industrial solvents and other organic compounds at industrial sites may result from weathering, chemical corrosion and accidental damage to pipes, storage vessels, processing equipment, transport vehicles, etc ., resulting in contamination. Improper disposal and/or negligent transport or storage of these chemicals has also contributed to the release of organic compounds into the environment at many locations in the United States and abroad, resulting in the contamination of substantial quantities of soil and groundwater ( e.g ., at former factories and industrial sites). This contamination gives rise to public health concerns, potential legal liability, and may cause extensive and long-term damage to the local ecosystem by accumulating in the tissue of animals and plants, resulting in mutation or death. In extreme cases, contaminated sites may be entirely unsuitable for human habitation without remediation.

[0005] Conventional technologies used to remediate gasoline and petroleum-contaminated soil and groundwater, include, for example: permanent removal of the contaminated soil to a secure landfill, incineration, indirect thermal treatment, aeration, pumping and treatment of groundwater, and soil vapor extraction with air-sparging. Removal of contaminated soil to landfills may be cost- prohibitive due to the high costs association with excavation, transportation and disposal of the contaminated soil. Moreover, there has been a recent trend towards environmental protection laws that disfavor such practices. Aeration, venting and soil vapor extraction with air-sparging may be used to increase the oxygen content in contaminated soils. Soil vapor extraction techniques create a vacuum in the soil which removes vapor within the pore space of contaminated soil, while air- sparging pulses air into the subsurface using air compressors. The increased oxygenation resulting from these processes may increase the rate of biodegradation of at least some contaminants by stimulating the growth of local aerobic bacteria at the site. [0006] Effective removal of hazardous materials by volatilization is slow and generally limited to contaminants that have a relatively high vapor pressure (e.g., generally reducing the effectiveness of this technique on polycyclic aromatic hydrocarbons). Venting and soil vapor extraction with air-sparging may increase the rate of degradation, but at an additional expense. Accordingly, conventional technologies currently being used for remediation of contaminated sites are often are expensive, time-consuming, and not always effective

Summary of Various Embodiments

[0007] In a general aspect, the present disclosure relates to novel combinations of Bacillus strains capable of bioremediating contaminated sites, compositions comprising these novel combinations and methods of using the same.

[0008] In a general aspect, the disclosure provides a composition comprising the following 11 Bacillus strains: B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis MO 17, 1E9 (PTA- 121389); B. licheniformis 07SI, 1E9; B. amyloliquefaciens 2E9 (PTA-121388); B. pumilus 1875, 1E9 (NRRL-1875); B. pumilus 07SI, 1E9; B. laterosporus CM33 2E9 (PTA-3592); B. laterosporus CM3 (PTA-3593); B. subtilis 10SI, 1E9; B. subtilis 1109, 1E9 (ATCC-9524); and B. mojavensis 1E8. In other aspects, compositions may comprise any combination of one or more of the 11 above-identified strains.

[0009] In some aspects, compositions according to the disclosure may comprise at least l x lO 3 , l x lO 4 , l x lO 5 , I c IO 6 , I c IO 7 , I c IO 8 , I c IO 9 , I c IO 10 , l x lO 11 , or l x lO 12 CFU/ml of the Bacillus strains described herein. Such compositions may comprise live cells, endospores, or a mixture thereof that have been spray-dried or lyophilized. In some aspects, compositions may comprise additional components such as a nutrient source (e.g., approximately 1, 2, 3, 4 or 5 wt% of a sugar capable of being metabolized by the blend of Bacillus cells or endospores), or chemical additives that function to bioremediate a contaminant (e.g., one or more enzymes, solubilizers, or surfactants). In other aspects, compositions may comprise one or more of the Bacillus strains described herein without any such chemical additives.

[0010] Compositions described herein may be capable of or adapted to bioremediate a contaminant comprising one or more of the following: an aliphatic or aromatic hydrocarbon; a polycyclic aromatic hydrocarbon (PAH); a substituted hydrocarbon; a halogenated or oxygenated hydrocarbon; an organic compound; a volatile organic compound (VOC); a semi-volatile organic compound (SVOC); and/or benzene, toluene, ethylbenzene or xylene. Some compositions described herein may be capable of reducing an amount or concentration of a contaminant by at least 50%, 60%, 70%, 80%, 90%, 95% or 99% within 30, 60, 90 or 120 days after being applied to an area that contains the contaminant.

[0011] In a general aspect, a method for bioremediating a contaminated area according to the disclosure may comprise: (i) treating the contaminated area by applying a plurality of Bacillus cells or endospores, wherein the plurality of Bacillus cells or endospores comprise one or more Bacillus strains; and (ii) reducing an amount or concentration of at least one contaminant (e.g., an aliphatic or aromatic hydrocarbon) in the area, wherein the one or more bacteria is/are B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis M017, 1E9 (PTA-121389); B. licheniformis 07SI, 1E9; B. amyloliquefaciens 2E9 (PTA-121388); B. pumilus 1875, 1E9 (NRRL- 1875); B. pumilus 07 SI, 1 E9; B. laterosporus CM33 2E9 (PTA-3592); B. laterosporus CM3 (PTA- 3593); B. subtilis 10SI, 1E9; B. subtilis 1109, 1E9 (ATCC-9524); and B. mojavensis 1E8. The contaminated area may include soil, gravel, concrete, pavement, surface water, and/or groundwater containing at least one contaminant, such as a hydrocarbon. [0012] In some aspects, the plurality of Bacillus cells or endospores are applied as a single blended composition. In other aspects, a composition may comprise one or more of the above- identified strains (e.g., any individual strain or a blend of any 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these strains). In some exemplar} ' aspects, the plurality of Bacillus cells or endospores are applied to the contaminated area (e.g., hydrocarbon-contaminated soil or groundwater): (a) as a single blended composition; or (b) separately, as one or more compositions applied simultaneously or in sequence. In either case, the plurality of Bacillus cells or endospores may he applied to the contaminated soil or groundwater: (a) as a slum ; (b) suspended in a liquid formulation; (c) as a spray-dried formulation; and/or (d) as a powder. In some exemplary aspects, two or more of the plurality of Bacillus cells or endospores in the single blended composition are present in a synergistically effective amount with respect to reducing an amount or concentration of at least one hydrocarbon contaminant in the contaminated site. In other aspects, the compositions may comprise live or spray-dried cells or endospores, as is desired for a given application.

[0013] In some aspects, the contaminant may comprise one or more of the following: (a) benzene, ethanol, ethylbenzene, isopropylbenzene, isopropyl ether, //-butylbenzene, //- propylbenzene, .vfc-butyl benzene, methyl /-butyl ether (MTBE), toluene, ethylbenzene, xylene, naphthalene, l,2,4-trimethylbenzene, 1,3,5 trimethylbenzene, a chlorinated hydrocarbon, and/or a polycyclic aromatic hydrocarbon (PAH); (b) total benzene, toluene, ethylbenzene, xylenes (total “BTEX”); (c) total xylenes; (d) total petroleum hydrocarbons as gasoline (TPH-G); and/or (e) total petroleum hydrocarbons as diesel (TPH-D), total petroleum hydrocarbons as oil and grease (TPH- O&G); and/or a semi-volatile organic compound. In exemplary aspects, following treatment, the concentration or amount of at least one contaminant in the contaminated are may be reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 99% as measured after 30, 60, 90 or 120 days compared to an untreated sample of the contaminated area.

[0014] Various compositions comprising these Bacillus combinations and methods of using the same are described herein. Additional aspects will be readily apparent to one of skill in light of the totality of the disclosure.

Brief Description of the Drawings

[0015] FIG. 1 is a table summarizing the bioremediation results of Example 3.

[0016] FIG. 2A is a graph illustrating the total bacterial abundance (CFU/g, wet weight) in soil treated with Bio-Rem at Ox, 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30- day period.

[0017] FIG. 2B is a graph illustrating the abundance of the bacterial species included in Bio- Rem (CFU/g, wet weight) in soil treated with Bio-Rem at 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period.

[0018] FIG. 2C is a graph illustrating the relative concentration (CFU/lxCFU) of bacteria in soil treated with Bio-Rem at 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. Concentrations for the 0.25x, 0.5x and 2x samples are shown normalized against the concentration of the lx sample at each time-point.

[0019] FIG. 3 is a graph illustrating the results of a qualitative odor ranking assay, which ranked the odor of sites treated with Bio-Rem at Ox, 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. A higher odor ranking indicates that a stronger odor was detected. Detailed Description of Various Embodiments

[0020] The disclosure provides compositions comprising combinations of Bacillus bacteria capable of biodegrading various organic compounds (e.g., aliphatic and aromatic hydrocarbons, substituted hydrocarbons, halogenated hydrocarbons, polycyclic aromatic hydrocarbons, volatile and semi-volatile organic compounds), and methods of bioremediation where such compositions are applied to contaminated sites. As described in further detail below, combinations of Bacillus bacteria described herein have effectively bioremediated contaminants (e.g., benzene, ethylbenzene, xylene and toluene) at various sites, with contaminant reductions of 90% or more observed in some instances.

[0021] In a general aspect, a composition according to the disclosure may comprise a blend of cells or endospores from multiple Bacillus strains, wherein the strains comprise: B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis M017, 1E9 (PTA-121389); B. licheniformis 07SI, 1E9; B. amyloliquefaciens 2E9 (PTA-121388); B. pumilus 1875, 1E9 (NRRL-1875); B. pumilus 07SI, 1E9; B. laterosporus CM33 2E9 (PTA-3592); B. laterosporus CM3 (PTA-3593); B. subtilis 10SI, 1E9; B. subtilis 1109, 1E9 (ATCC-9524); and B. mojavensis 1E8. It is understood that the strain accession numbers identified above indicate that such sequences are deposited with, and available from, the American Type Culture Collection (“ATCC” and“PTA” accession numbers) or the ARS Culture Collection (“NRRL” accession numbers). Compositions comprising this specific blend of 11 Bacillus strains are referred to herein as“Bio-Rem.” In other alternative aspects, a composition may comprise any subset of the 11 Bacillus strains found in Bio-Rem. For example, a composition according to the disclosure may comprise cells and/or endospores from at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the Bacillus strains identified above. Moreover, the relative concentration of the cells and/or endospores in such compositions may be equivalent or enriched as to one or more of the included strains (e.g., a composition may comprise a subset of 1, 2, 3, 4, or 5 of the strains identified above wherein the cells and/or endospores for each strain are present in the mixture at approximately equivalent concentrations).

[0022] In some exemplar} ' aspects, a composition according to the disclosure may comprise a mixture of four or more Bacillus strains, comprising at least one B. licheniformis , B. subtilis, B. amyloliquefaciens , and B. pumilus , strain identified herein. In some aspects, the at least one B. licheniformis strain comprises one or more of B. licheniformis 1001, 1E9 (ATCC-l 1946); B. licheniformis M017, 1E9 (PTA-121389); and/or . licheniformis 07SI, 1E9. In some aspects, the B. amyloliquefaciens strain comprises /? amyloliquefaciens 2E9 (PTA-121388). In some aspects, the B. subtilis strain comprises one or more of B. subtilis 10SI, 1E9; and/or . subtilis 1109, 1E9 (ATCC-9524). In some aspects, the B. pumilus strain comprises B. pumilus 07SI, 1E9. It is understood that any of the compositions or methods described herein may comprise (or use) any combination of the Bacillus strains described in this passage (e.g., any combination of 4, 5, 6 or 7 of these Bacillus strains).

[0023] A composition according to the disclosure (e.g., Bio-Rem) may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, or 9x l0 10 colony forming units per mL (CFU/mL). A colony forming unit (CFET) is an estimation of the total population of viable cells capable of growing and replicating giving rise to a single colony. This estimation is based upon the assumption that a single cell (or spore) gives rise to a single colony— thus a colony forming unit. Because spores will germinate, grow, and replicate on solid media, CFETs can be an estimate of viable cell or spores. CFETs in the total population may be estimated by serially diluting the given culture or solution and evenly spreading a single dilution on a solid complex medium such as Tryptic Soy Agar (TSA) and incubating at 37 °C. overnight. The number of colonies which grow overnight multiplied by the total dilution factor will give the number of CFUs/mL, an estimate of the number of viable spores and/or cells/mL. Spore estimates may be done in the same protocol with the added step of holding the dilution at 80 °C for 5 minutes before spreading on solid media; this step ensures all vegetative cells are killed. Spores, which are able to withstand high heat, remain unharmed during this 80 °C. incubation. When a composition comprises more than one Bacillus strain, as in the case of Bio- Rem, the same protocol may be used, and the concentration of individual strains may be determined from their distinct and differentiable colony morphology.

[0024] The CFU/ml of each Bacillus strain in a composition according to the disclosure may vary from 1 x 10 3 CFU/ml up to 1 x 10 12 CFU/ml. In some aspects, such compositions may comprise at least U 10 3 , U lO 4 , U lO 5 , U lO 6 , U lO 7 , U 10 8 , U 10 9 , U lO 10 , U lO 11 , or U lO 12 CFU/ml or an average number of CFU/ml within this range for any or all of the strains in the composition. In some aspects, the concentration for any or all of the bacteria may comprise a range wherein the minimum and maximum amounts are selected from any of the above-identified values (e.g., 1 x 10 8 to U lO 12 CFU/ml, l x l0 9 to U 10 12 CFU/ml, l x l0 10 to U lO 12 CFU/ml).

[0025] Compositions described herein may comprise live cells or endospores. Endospores may be preferable for compositions intended for long-term storage. Endospores may be obtained by culturing Bacillus cells from each strain in the desired composition according to methods well known in the art. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, and liquid surface culture. Towards the end of fermentation, as nutrients are depleted, Bacillus cells begin the transition from growth phase to sporulation phase. The bacterial spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential -flow filtration, depth filtration, and evaporation. Compositions comprising endospores (or live cells) according to the present disclosure may be spray-dried or freeze-dried.

[0026] In some aspects, compositions comprising one or more of the Bacillus bacteria described herein are the only active agent for bioremediation. Such compositions may comprise other agents that do not function to bioremediate a contaminant (e.g., nutrients capable of being metabolized by the Bacillus strains). In other aspects, compositions may comprise one or more of the Bacillus strains described herein and at least one chemical and/or biological additive that functions to bioremediate a contaminant (e.g., enzymes, solubilizers or surfactants).

[0027] In some exemplary aspects, the contaminant that may be bioremediated using a composition according to the disclosure comprises a semi-volatile organic compound (SVOC) or a polycyclic aromatic hydrocarbon (PAH). Exemplary SVOCs and PAHs include, without limitation: acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(g,h,i)perylene, benzo(k)fluoranthene, 4-bromophenylphenyl ether, butylbenzylphthalate, 4-chloro-3-methylphenol, bis(2-chloroethoxy)methane, bis(2-chloroethyl) ether, 2-chloronaphthalene, 2-chlorophenol, 4-chlorophenylphenyl ether, chrysene, dibenz(a,h)anthracene, 3,3'-dichlorobenzidine, 2,4-dichlorophenol, di ethyl phthal ate, 2,4- dimethylphenol, dimethylphthalate, di-n-butylphthalate, 4,6-dinitro-2-methylphenol, 2,4- dinitrophenol, 2,4-dinitrotoluene, 2,6-dinitrotoluene, di-n-octylphthalate, bis(2- ethylhexyl)phthalate, fluoranthene, fluorene, hexachloro- 1,3 -butadiene, hexachlorobenzene, hexachlorocyclopentadiene, hexachloroethane, indeno(l,2,3-cd)pyrene, isophorone, naphthalene, nitrobenzene, 2-nitrophenol, 4-nitrophenol, n-nitrosodimethyl amine, n-nitroso-di-n-propylamine, n-nitrosodiphenylamine, 2,2'-oxybis(l-chloropropane), pentachlorophenol, phenanthrene, phenol, pyrene, 1, 2, 4-tri chlorobenzene, and 2,4,6-trichlorophenol. [0028] In some aspects, the contaminant to be bioremediated may comprise a volatile organic compound (VOC). Exemplary VOCs include, without limitation: petroleum hydrocarbons (e.g., benzene, bromobenzene, bromoform, bromomethane, n-butylbenzene, sec-butylbenzene, tert- butylbenzene, l,2-dibromoethane (EDB), dibromomethane, diisopropyl ether, ethylbenzene, hexachloro-l, 3-butadiene, isopropylbenzene (cumene), methylene chloride, methyl-tert-butyl ether, naphthalene, n-propylbenzene, styrene, toluene, l,2,4-trimethylbenzene, 1,3,5- trimethylbenzene, o-xylene, m-xylene, p-xylene) and chlorinated compounds (e.g., bromochloromethane, bromodichloromethane, chlorobenzene, chloroethane, chloroform, chlorom ethane, l,2-dibromo-3-chloropropane, dibromochlorom ethane, 1 ,2-di chlorobenzene, 1,3- dichlorobenzene, l,4-di chlorobenzene, dichlorodifluoromethane, l,2-dichloropropane, 1,3- dichloropropane, 2,2-dichloropropane, l, l-dichloropropene, cis-l,3-dichloropropene, trans-l,3- dichloropropene, 2-chlorotoluene, 4-chlorotoluene, carbon tetrachloride, 1, 1 -di chloroethane, 1 ,2- di chloroethane, l,l-dichloroethene, cis-l,2-dichloroethene, trans-l,2-dichloroethene, tetrachloroethene, 1,2, 3-trichlorobenzene, 1, 2, 4-tri chlorobenzene, trichlorofluoromethane, 1,2,3- trichloropropane, 1,1,1 -tri chloroethane, 1 , 1 ,2-tri chloroethane, l, l,l,2-tetrachloroethane, 1, 1,2,2- tetrachloroethane, trichloroethene, and vinyl chloride).

[0029] Contaminants that may be bioremediated using compositions of the present disclosure further include, without limitation, various organic compounds such as aliphatic hydrocarbons (e.g., C5-C36), aromatic hydrocarbons (e.g., C9-C22), substituted hydrocarbons (e.g., chlorinated hydrocarbons). Specific petroleum distillates and derivatives that may be bioremediated include crude oil, refined oil, fuel oils, diesel oils, gasoline, hydraulic oils, motor oil, and kerosene, as well as individual components thereof. As exemplified by the examples described in detail below, volatile aromatic hydrocarbons such as benzene, toluene, ethylbenzene and xylenes (BTEX) may be efficiently degraded by the present compositions. Trimethylbenzenes, and other PAHs such as naphthalene, anthracene, acenaphthene, acenaphthylene, benzo (a) anthracene, benzo (a) pyrene, benzo (b) fluoranthene, benzo (g,h,i) perylene, benzo (k) fluoranthene and pyrene, are also common constituents of fuel oils and heavier petroleum compounds and may be degraded using compositions and methods according to the present disclosure.

[0030] Petroleum derivatives are commonly associated with other pollutants such as chlorinated aliphatics, chlorinated aromatics and non-chlorinated aromatics. Other chlorinates hydrocarbons include methylene chloride, l, l-dichloroethane, chloroform, l,2-dichloropropane, dibromochlorom ethane, l, l,2-trichloroethane, 2-chloroethylvinyl ether, tetrachloroethene (PCE), chlorobenzene, l,2-dichloroethane, l,l, l-trichloroethane, bromodichloromethane, trans-l,3- dichloropropene, cis-l,3-dichloropropene, bromoform, chloromethane, bromomethane, vinyl chloride, chloroethane, l,l-dichloroethene, trans-l,2-dichloroethene, trichloroethene (TCE), dichlorobenzenes, cis-l,2-dichloroethene, dibromomethane, l,4-dichlorobutane, 1,2,3- trichloropropane, bromochlorom ethane, 2,2-dichloropropane, l,2-dibromoethane, 1,3- dichloropropane, bromobenzene, chlorotoluenes, tri chlorobenzenes, trans-l,4-dichloro-2-butene and butylbenzenes. Bacillus combinations and bioremediation methods described herein may also be used to degrade one or more of these compounds.

[0031] In some aspects, the Bacillus combinations and methods described herein may be used to biodegrade chlorinated aliphatic hydrocarbons, such as l,l,2-trichlorethylene (TCE). Other chlorinated aliphatic hydrocarbons that may be degraded include tetrachloroethylene (PCE), 1, 1,1- tri chloroethane, l,2-dichloroethylene (DCE), and vinyl chloride (VC).

[0032] Methods for bioremediating a contaminated area are also provided. A method for bioremediating a contaminated area (e.g., hydrocarbon-contaminated soil or groundwater) may comprise: (i) treating the contaminated area by applying a plurality of Bacillus cells or endospores; and (ii) reducing an amount or concentration of at least one contaminant in the area; wherein the plurality of Bacillus cells or endospores comprise any combination of Bacillus strains described herein. In some aspects, the plurality of Bacillus cells or endospores are applied is Bio-Rem. In other aspects, a composition may comprise one or more of the Bacillus strains in Bio-Rem (e.g., any individual strain in Bio-Rem or a combination of any 2, 3, 4, 5, 6, 7, 8, 9, 10 of the 11 strains in Bio-Rem).

[0033] Compositions of Bacillus strains according to the disclosure may display synergistic effects whereby the biodegradation of one or more contaminants (e.g., aliphatic, aromatic, polycyclic aromatic and chlorinated hydrocarbons) in the treated area proceeds at a higher rate compared to samples treated with a single or subset of Bacillus strains in the combination. In these instances, the observed results are greater than would be expected based on their individual capacities for metabolizing such compounds (i.e., greater than additive).

[0034] As a result, compositions comprising combinations of Bacillus strains according to the disclosure may therefore provide a broadly applicable solution for contaminant (e.g., hydrocarbon, PAH and/or VOC) bioremediation at various sites that cannot be efficiently remediated using conventional methods, which may be costly, slow or reliant on indigenous bacteria for bioremediation not present at the site in effective quantities (e.g., necessary indigenous bacteria may be unable to colonize contaminated soil or groundwater at the site due to the absence of a required nutrient or an unsuitable physical/biochemical environment).

[0035] As illustrated by the examples provided herein, compositions according to the disclosure may be easily applied to contaminated sites. The bacterial compositions may be applied, for example, as a slurry directly to a contaminated above-ground area or as an inoculant applied to groundwater using a vent. Repeated administrations may be desirable to maintain colonization at the site and to enhance rate of bioremediation. However, in some aspects a single application may be sufficient. In either case, application of Bacillus combinations according to the present disclosure is often faster and may be substantially less expensive than conventional techniques (e.g., which may require costly excavation or vacuum/aeration equipment). Moreover, Bacillus combinations according to the present disclosure may be assayed to determine a biodegradation profile prior to application in order to prepare a combined composition particularly suitable for degradation of the specific compounds present at the contaminated site.

[0036] Bacillus combinations according to the disclosure may be applied to a contaminated directly to an above-ground surface (e.g., as a slurry) or to a subsurface area (e.g., via a well, vent, excavation, or pipe). In some aspects, the Bacillus compositions comprise spore-forming strains. Compositions comprising endospores produced by these Bacillus strains may be uses in some applications. Similarly, spray-dried compositions of Bacillus cells or endospores may be particularly suitable for some applications where long-term viability is desirable.

[0037] Bacillus combinations described herein may be used to bioremediate soil, groundwater and other environments contaminated with various petroleum constituents. While particular attention is drawn to the use of the disclosed combinations to bioremediate soil and groundwater, other suitable environments include but are not limited to, sediment, sand, gravel, groundwater, aquifer material, surface water, and landfills. In still further aspects, Bacillus combinations described herein may be used to biodegrade contaminants extracted from a contaminated site. For example, contaminated soil may be slurried or leached with water (the leachate) in order to remove contaminants. This leachate may then be brought into contact with an effective amount of one of the Bacillus combinations described herein (e.g., a liquid culture grown in a bioreactor with a carbon source), resulting in biodegradation of at least some of the contaminants.

[0038] The present methods may be used to perform bioremediation on the surface and/or underground sites. In instances where contamination occurs underground (e.g., in the case of contaminated groundwater), Bacillus combinations according to the present disclosure may be applied to the contaminated area using a well, injection tools, pipe, or other delivery system. In some aspects, suspensions containing a Bacillus combination may be delivered to the site of contamination (e.g., a hydrocarbon plume from an oil leak in proximity to a groundwater aquifer), alone or in conjunction with other agents (e.g. a nutrient source to stimulate growth of the Bacillus cells after delivery) and/or chemical agents (e.g., enzymes, surfactants, solublizers) which may help solubilize contaminants and increase the rate of degradation.

[0039] The following non-limiting examples are provided to further illustrate the embodiments disclosed herein.

EXAMPLES

Example #1: Laboratory Treatment and Analysis of Petroleum Contaminated Groundwater

[0040] The effectiveness of Bio-Rem (an 11 -strain blend of Bacillus cells or endospores described herein) on petroleum-contaminated groundwater was tested. Groundwater samples were collected in the field from a petroleum impacted site and immediately delivered to an independent laboratory for analysis and treatment. The laboratory decanted the groundwater sample into 1,000 ml beakers and divided the sample into untreated (control sample) and treated samples. Bio-Rem was then applied to the samples selected for treatment at a concentration of 0.5 g/L of groundwater. The untreated and treated samples were maintained under laboratory conditions for 30 days before being analyzed to determine the residual amount of volatile organic compounds in both samples. [0041] As summarized in Table 1 below, laboratory tests indicated that Bio-Rem significantly degraded (>92% decrease in contaminant levels) petroleum-derived compounds in the groundwater sample under laboratory conditions.

[0042] Table 1. Summary of Groundwater Analytical Results

Example #2: On-Site Treatment of Soil Contaminated with Heating Oil

[0043] In an effort to evaluate the effectiveness of Bio-Rem on petroleum contaminated soil, Bio-Rem was applied to a surface spill of heating oil that occurred within a residence basement. Using hand tools, researchers excavated an approximately 24” x 24” x 20” area of contaminated soil within the basement around the perimeter of the slab foundation of a residence. This area was sampled (collection 1) and analyzed for Total Petroleum Hydrocarbons as Diesel (TPH-D) and Gasoline (TPH-G).

[0044] Following sampling, approximately 140 g of Bio-Rem was mixed with approximately 2.5 gallons of water and applied to the contaminated area. An untreated, control area was also disturbed and sampled per above. Confirmatory sampling was performed approximately six weeks later (collection 2) and laboratory analyses indicated that the TPH-G in the treated and control areas had decreased at similar rates. The TPH-D concentrations remained essentially unchanged for the treated and control areas. However, a perceptible odor change was noted in the two areas treated with Bio-Rem and laboratory testing indicated that there had been a significant change in the associated chromatogram which included peak reduction and retention time changes. A soil sample from one of the treated areas was collected approximately 12 weeks following application of the product (collection 3) and indicated a 72% decrease from the initial concentration.

[0045] As a result, researchers collected soil from the contaminated area for additional ex-situ treatment and analysis. An additional 30 grams of Bio-Rem, 5 grams sugar, and one liter of water were added to the sample, as a second treatment dose. Researchers collected and tested a soil sample for TPH-D approximately nine weeks later (collection 4). The sample exhibited an approximately 81.9% decrease in TPH-D since the second sample collection, with an approximately 35.1% decrease since the third sample collection.

[0046] Table 2. Summary of Soil Analytical Results

[0047] As illustrated by Table 2, a reduction of approximately 82% TPH-D (i.e., 4,140 mg/kg to 753 mg/kg) was observed following two treatments of the contaminated soil with Bio-Rem over a span of approximately 4 months. Example #3: On-Site Treatment of Petroleum Contaminated Groundwater Monitoring Well

[0048] Bio-Rem was field tested at a petroleum contaminated groundwater site in North Carolina. The site is a gasoline station located within the coastal plain physiographic province with sandy soils and relatively shallow groundwater (i.e., approximately 5-6 feet below ground surface). An on-site monitoring well has exhibited persistent dissolved-phase petroleum groundwater contamination. One kilogram of Bio-Rem was introduced into the monitoring well via suspension within a five-foot, one inch Schedule 40 PVC pipe with 0.10 slotted screen. A small amount of sugar (approximately 1-2% by weight) was added in order to stimulate microbial activity. The goal was to introduce microbes into the contaminant plume to facilitate petroleum degradation.

[0049] Groundwater samples were collected from the monitoring well prior to and, at approximate 30-day intervals, following injection activities in order to evaluate the effectiveness of Bio-Rem on dissolved-phase petroleum constituents and influence on bacteriological populations under field conditions. The groundwater samples were then analyzed for volatile organic compounds (VOCs) by Standard Method 6200B including ethanol, methyl tert-butyl ether (MTBE), and isopropyl ether (IPE). In addition, researchers collected groundwater samples for analysis via heterotrophic plate count (HPC). This procedure is used for estimating the number of live culturable heterotrophic bacteria in water. Colonies may arise from pairs, chains, clusters, or single cells, all of which are included in the term“colony-forming units” (CFET).

[0050] Subsequent sampling and analysis of the Bio-Rem treated monitoring well indicated that, after 60 days following the initial Bio-Rem injection: (1) Benzene decreased 64%; (2) Benzene, toluene, ethylbenzene, and total xylenes (BTEX) decreased 72%; and (3) methyl tert- butyl ether (MTBE) decreased 70%. In addition, CFETs have increased from 130, prior to treatment with Bio-Rem, to as high as 186,700,000. Results from this study are summarized in Table 3, which is provided as FIG. 1.

[0051] As illustrated by FIG. 1, a substantial reduction in the concentration of various contaminants was observed within 60 days of the initial treatment (i.e., the 4/10/17 sample). These results confirm Bio-Rem’s effectiveness for short-term bioremediation. Long-term results from this study illustrate a trend towards further reduction for most of the analyzed compounds. By the most recent collection date (9/13/17), approximately seven months after the additional treatment, the concentration of benzene in the sample had improved by 78%. The levels of other notable hydrocarbon contaminants were also further reduced, with l,2-dichloroethane, I-Propylbenzene, n-Butylbenzene, n-Propylbenzene, l,2,4-Trimethylbenzene, MTBE and other contaminants showing an improvement of over 90% compared to the original sample.

[0052] In sum, Table 3 confirms that Bio-Rem is broadly useful as a bioremediation agent for reducing levels of hydrocarbons, PAH and VOC contaminants associated with petroleum. As indicated above, Bio-Rem is an exemplary Bacillus combination according to the disclosure and thus the results of this study provide further support for the use of other similar Bacillus combinations described herein for bioremediation of these and other petroleum-based contaminants.

Example #4: On-Site Treatment of Surface Spill of Mineral Oil

[0053] Bio-Rem was evaluated for effectiveness as a bioremediation agent for mineral oil following a lightning strike that resulted in the release of approximately 300 gallons of mineral oil at an electrical substation. Researchers applied approximately 750 gallons of an Bio-Rem slurry to the affected area. The site conditions, including energized electrical equipment and the need for uninterrupted facility operations, precluded the use of more traditional remedial methods such as over-excavation of contaminated soils.

[0054] The site measures approximately 750 square feet and is characterized by an approximately four-inch gravel layer underlain by clayey silts and silty sands soils. The application procedure included the on-site mixing of 16 kg of Bio-Rem (and sugar) in three approximate 250-gallon batches. The Bio-Rem was mixed with water using a high shear mixer and added to an on-site 275-gallon tote. The slurry was applied directly to the contaminated area (three applications) via a fabricated series of perforated pipes and a gravity fed two-inch hose. Subsequent sampling of the soil in the source area indicated a decrease in Total Petroleum Hydrocarbons as Diesel (TPH-D) of nearly 50% within four weeks of application. At twelve weeks following the application of Bio-Rem, the soil samples generally exhibited an average decrease of approximately 76% in TPH-D concentrations, with the soil sample collected in the immediate source area of the release exhibiting an 87% reduction in TPH-D concentrations.

[0055] Results from this study further illustrate the broad usefulness of Bio-Rem as a bioremediation agent for mineral oil spills.

Example #5: Bacterial Abundance and Odor Reduction Studies

[0056] Studies were conducted to measure the concentration of total bacteria and Bio-Rem bacteria present in soil at sites treated with Bio-Rem at concentrations of 0.25x, 0.5x, lx and 2x compared to a control (i.e.,“Ox”), over a 30 day period. Hydrocarbon levels (e.g., PAHs) in the soil of treated and control sites were measured using Gas chromatography-mass spectrometry (GCMS). Treated sites were also subjected to a qualitative odor ranking assay in order to determine the relative effect of different Bio-Rem concentrations on odor reduction. The results of the these studies are summarized by FIGS 2A, 2B, 2C, and 3. [0057] FIG. 2A is a graph illustrating the total bacterial abundance (CFU/g, wet weight) in soil treated with Bio-Rem at 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. As illustrated by this graph, soil treated with Bio-Rem at any of these four concentrations displayed an approximately lOOx greater concentration of bacteria than the control sample (i.e., “Ox”) at all time-points. This increased concentration was maintained at a stable level across the 30-day period of this experiment, indicating that Bio-Rem treatment resulted in stable colonization of the treated sites.

[0058] FIG. 2B is a graph illustrating the abundance of the bacterial species included in Bio- Rem (CFU/g, wet weight) in soil treated with Bio-Rem at 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. As illustrated by this graph, treatment with Bio-Rem at 0.25x, 0.5x, lx or 2x concentrations resulted in stable colonization over the 30-day period, with the more concentrated treatments resulting in a higher steady-state level (e.g., the period spanning from day 7 to day 30).

[0059] FIG. 2C is a graph illustrating the relative concentration (CFU/lxCFU) of bacteria in soil treated with Bio-Rem at 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. Concentrations for the 0.25x, 0.5x and 2x samples are shown normalized against the concentration of the lx sample at each time-point. This graph illustrates the same dataset shown in FIG. 2B, reformatted to highlight the change in relative concentration of Bio-Rem bacteria present in soil treated at alternative concentrations (i.e., 0.25x, 0.5x and 2x). As illustrated by this graph, the 0.25x-treated soil samples were found to contain approximately 70% as many CFUs as the lx-treated soil. The 0.5x-treated soil samples initially displayed a comparable concentration of CFUs. However, the concentration of Bio-Rem bacteria in these samples increased over time, with a final concentration close to 95% of the concentration observed in the lx-treated soil samples by day 30. As illustrated by this graph, the concentration of Bio-Rem bacteria in the tested samples did not scale 100% with the treatment dose. However, the increased concentration scaled in a consistent manner.

[0060] FIG. 3 is a graph illustrating the results of a qualitative odor ranking assay, which ranked the odor of sites treated with Bio-Rem at Ox, 0.25x, 0.5x, lx or 2x, measured at various time-points over a 30-day period. A higher odor ranking indicates that a stronger odor was detected. As illustrated by this graph, higher Bio-Rem treatment concentrations resulted in a more pronounced decrease in detected odors, with noticeable differences at days 15 and 30 of the 30- day study.