WO2016196680A1 - Biofilm control and frack fluid production using bacillus subtilis activated with gfak - Google Patents

Abstract

A composition suitable for addition to frack water comprising: a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl in 50mM-Tris / HCl buffer, pH 7.5 (GFAK); wherein on germination the bacteria produce prodigious amounts of the natural biosurfactant surfactin, biocidal concentrations of subtilin and lanthionine-containing peptide antibiotics which act to competitively exclude unwanted bacteria; in addition they produce α-amylase enzymes which detach biofilm exopolysaccharide components; when added to fracking fluids they increase efficiency, control biofilm formation and reduce or remove the need to add environmentally-damaging chemicals to frack fluids.

Description

BIOFILM CONTROL AND FRACK FLUID PRODUCTION USING

BACILLUS SUBTLLLS ACTIVATED WITH GFAK

[0001] CROSS REFERNCE TO RELATED APPLICATION

[0002] This application claims the benefit of U.S. Provisional Application Serial No.

62/169,011, filed June 1, 2015, the disclosure content of which is hereby incorporated by reference in its entirety.

[0003] FIELD OF THE INVENTION

[0004] The invention relates to biofilm control, surfactant production and downstream bioremediation of fluids suited for secondary oil and gas recovery, and control and remediation of paraffin compound build-up in frack wells.

[0005] BACKGROUND OF THE INVENTION

[0006] The fracking industry utilizes water treated with chemicals and sand, which are injected into a wellbore under high pressure to extend and or create fractures in an oil or gas formation. The sand props open the fracture and provides a pathway for the oil or gas to flow out of the formation. Fracturing fluids consist of potable or non-potable water, a thickening agent to create a gel-like viscosity, bactericides, friction reducers, corrosion inhibitors, and salts such as calcium chloride.

[0007] Biocides are used to either kill or prevent the formation of bacteria that can cause corrosion, plug the well, or create sulfide reducing bacteria (SRB). Typical oilfield biocides are either oxidizing or non-oxidizing.

[0008] Indeed, SRB, and other bacteria form biofilms on surfaces. Biofilms are common in nature and are often associated with unwanted biofouling. Problems caused by biofilms include the ability of Legionella to persist in cooling towers and other water systems due to its complex interrelationships with other microflora (Murga et al, 2001), fouling of oil and gas pipelines and ships' hulls, dental plaque causing tooth decay and microbial colonisation of indwelling or percutaneous medical devices such as catheters, artificial valves, j oints, stents etc.

[0009] Classical microbiological studies have concentrated on bacteria in their planktonic state - single organisms or small groups suspended in aqueous culture - but most bacteria in nature colonise natural and man-made surfaces using exogenous polysaccharides (the glycocalyx) for adhesion, then multiply to form colonies which grow into complex, sessile biofilms (Costerton et al., 1995).

[00010] Biofilms are complex, irregular, heterogeneous hydrated structures where a consortium of microcolonies of bacteria can divide freely (Lewandowski, 2000). Inhabiting a biofilm confers significant advantages on its resident bacteria. They are protected from environmental threats such as desiccation, antibiotics (Ishida et al, 1998) and biocides (McDonnell and Russell, 1999), and form symbiotic relationships with other residents allowing exchange of nutrients, metabolites and genetic material.

[00011] The glycocalyx is the principal component of the extracellular matrix (ECM) - which provides the biofilm' s structural integrity and is chemically similar to the bacterial capsule surrounding pathogens such as Streptococcus, Klebsiella, Pseudomonas and

Neisseria. The ECM is more diffuse and less distinct than a capsule and accounts for 50-90% of the organic carbon content of the biofilm (Flemming, Wingender and Mayer, 2000). It has been shown that B. subtilis biofilms are more non-wetting (that is, they have greater repellency to water) than Teflon® and are impervious even to 80% ethanol (Epstein et al, 2011), hence biofilms' recalcitrance.

[00012] Accordingly, control of the ECM and of the pathogenic biofilms is necessary to ameliorate or modify biofilms, surfactant production and modification of fluids in the gas and oil industry. [00013] SUMMARY OF THE INVENTION

[00014] The Invention controls biofilms that hamper secondary oil and gas recovery in tracking operations as well as producing biosurfactants to increase the efficiency of fracking processes. The Invention removes the need for synthetic chemical biocides in fracking operations and reduces or removes the need for the addition of chemical surfactants. The Invention also remediates waste waters reducing biological and chemical oxygen demand to levels where the wastes are safe to discharge.

[00015] The Invention comprises a blend of Bacillus species added to fracking water and activated with a blend of 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KC1 in 50mM-Tris / HC1 buffer, pH 7.5 (GFAK); on germination the bacteria produce prodigious amounts of the natural biosurfactant surfactin, biocidal concentrations of subtilin and lanthionine-containing peptide antibiotics which act to competitively exclude unwanted bacteria; in addition they produce a-amylase enzymes which detach biofilm exopolysaccharide components; when added to fracking fluids they increase efficiency, control biofilm formation and reduce or remove the need to add environmentally-damaging chemicals to frack fluids.

[00016] Description of the Invention

[00017] The impact of bacterial biofilms causing biofouling and corrosion in engineering applications is well documented. Accordingly, described herein is a biofilm control composition comprising one or more strains of Bacillus subtilis, which are prodigious producers of natural lantibiotics and other biocidal moieties, acting synergistically. Together, in combination with suitable surfactant provides a means for controlling biofilms in fracking applications with direct and indirect biocidal activity while reducing or removing the need for other frack fluids. [00018] Bacterial biofilms, like disease-causing microbes (including nosocomial pathogens) use biofilms as an ideal habitat for reproduction and exchange of genetic material via plasmids. It has now been shown that bacteria from different genera can exchange resistance plasmids, and this ability to cross the species barrier has led to the rapid global spread of many different bacteria becoming resistant to both common antibiotics and antibiotics of 'last resort'. This has already been found in healthcare facilities in the USA. In the industrial application, there is less of a concern of generation of bacteria that are resistant for human disease, but of generation of certain bacteria that have increased spoliation issues and cause greater damage in the gas and oil industry.

[00019] A particular issue with biofilms is that they are impervious to traditional chemical biocides and antibiotics and so cannot be controlled by conventional biocidal chemicals. Biofilms in nature often contain a variety of bacteria, algae and protozoa but it is possible to grow a biofilm with a single species. The Bacillus subtilis in the invention can readily switch from its motile, planktonic state to grow in bundled chains and then multicellular communities (Kearns et al, 2005). This ability to form complex structures gives B. subtilis the ability to form extremely robust biofilms, which can be used advantageously to generate and control benign biofilm formation on a surface.

[00020] This invention provides a means of modifying the microbial ecology of biofilms to exclude and destroy pathogens by replacing them with non-pathogenic bacteria. The invention uses a mix of bacteria that invade pathogenic biofilms to competitively exclude and destroy pathogenic bacteria. The introduced bacteria produce natural antibiotics to kill pathogenic bacteria and biosurfactants to degrade organic soiling. The antibiotics produced by the introduced bacteria are not suitable for use in treating human disease and so will not drive antibiotic resistance. [00021] This combination of biocidal activity against pathogenic bacteria is effective in removal of soiling and therefore offers a new tool for control of problemating bacteria in industrial applications, which are not reliant on traditional means that merely drive bacterial resistance, or require constant application of expensive and toxic compositions to oil and gas wells.

[00022] The Bactillus subtilis species used in the invention is not a human pathogen

(Osipova et al, 1998). The species utilized is a Gram-positive spore former and a facultative anaerobe (Nakano and Zuber, 1998) and is particularly well suited to this application due to its lack of pathogenicity, ubiquity in the environment (principally soil) and ease of culture.

[00023] We have demonstrated with the invention that it is possible to apply suitable strains of B. subtilis in the same way as traditional cleaning products and artificially introduce a biofilm that digests organic soils by producing a suitable suite of exogenous enzymes. The composition, upon mixing with water or another carrier, can be used in the same way as a traditional 'spray and wipe' cleaning product, namely, application to a surface or a cloth, and mechanically wiping the surface however it possesses powerful applications in the gas an oil industry through different application mechanisms. For example the composition is suitable for use in at least three primary oil and fracking applications including biofilm control in fracking water, frack fluid remediation, and paraffin or other wax removal from oil and gas pipes and materials. Accordingly, biofilms offer a new paradigm for control of problematic biofilm and bacteria; their ubiquity and recalcitrance is only now becoming apparent and the ability of the Invention to re-engineer existing biofilms and digest body fluids, fats, dirt, grease and grime as well as excluding pathogenic bacteria presents an environmentally responsible and effective solution for the oil and gas industry.

[00024] For the purposes of treatment of frac (or frack - used interchangeably) fluid, in a preferred embodiment, the composition is a bacteria reducing or eliminating product that is presented as a dry powder in its original form and mixed with sterile water to form a concentrate. The concentrate depends on the amount and type of bacteria being treated.

[00025] The composition can be mixed with frac water as a dry powder mixed in a hopper blender typical to all frac equipment. The product can also be pre-mixed in special tanks called frac tanks which hold 500 barrels of water (21,000 gallons) and are equipped with wheels for easy transport from well to well. The tank would be delivered to a well location, then filled with water, then the composition would be mixed into the tank and circulated for uniform distribution.

[00026] The composition can also be mixed and pumped during the frac treatment in a process called "on the fly" where a blender truck is taking in water, chemicals and sand and mixing these to prescribed formula and suppling this mixture to the pressure pumps for injection into the well.

[00027] Accordingly, a biofilm control composition comprises the use of one or more strains of Bacillus subtilis, which are prodigious producers of surfactin, lantibiotics and other biocidal moieties. The composition functions through several primary actions, including competitive exclusion, reduction of the native biofilm and quorum sensing, wherein present bacteria are converted from pathogenic to benign.

[00028] The competitive exclusion is generated as the bacteria provided in the composition are generally stronger, grow faster, and can out compete the pathogenic bacteria. Accordingly, application of these bacteria utilize the resources that pathogenic bacteria need to grow and flourish. As these resources are utilized and usurped by the benign bacteria, the pathogens are competitively excluded and begin to die out.

[00029] The dry powered composition is generated wherein the bacilli are sporulated at high temperatures in the presence of divalent cations to give greater resistance to heat and pressure than wild strains and are activated by high pressure or by the addition of GFAK. Indeed, because of the nature of the application into fracking fluids that may include high temperatures, high temperatures, or both, it is necessary to modify the Bacilli to remain active upon these conditions. The inclusion of GFAK into the composition provides for a mechanism to induce spore germination upon application with an appropriate buffer solution. Together, these components are mixed into frack fluids at an appropriate concentration and then utilized in the frack wells or in a frack fluid pond or holding container.

[00030] The dry powered composition may also be suitably provided in a concentrated aqueous form, or in a ready to use concentration for application into a water environment. In the case of a concentrated aqueous form, the composition is generated by forming a carrier composition and adding to that carrier spores from Bacillus subtilis, GFAK composition and a buffer.

[00031] The invention is also suitable for remediation of recovered fracking waters and reduces biological and chemical oxygen demand to levels rendering the waste safe to discharge. Furthermore, the compositions described herein provide for the artificial introduction of benign biofilms that offer a new paradigm for both fracking efficiency, biofilm control, remediation of waste frack fluids, and for paraffin removal.

[00032] The invention is a proprietary blend of naturally-occurring, non-GMO

'probiotic' bacteria designed to make hydraulic fracturing processes dramatically more efficient and less reliant on environmentally-harmful chemicals. The composition tested comprises an effective amount of B. subtilis strains selected from the group consisting of the following strains: 1A5, 1A28, 1A84, 1A122, 1A698, 1A699, 1A751, and 1A752, and combinations thereof to a concentration of lxlO⁸ CFU ml^"1. In further embodiments, any one of the 168 known strains of B. subtilis may be advantageously utilized based on the production of surfactin, subtilin and lanthionine-containing peptide antibiotics, and a-amylase enzymes suitable for detaching biofilm exopolysaccharide components. Because the bacteria continue to generate these components, there is no need to continue to add surfactin or other antibiotics to the frac water, on a continued basis. In turn, this can dramatically improve the nature of the outcoming frac waste water, such that the waste water requires less treatment than untreated waste water.

[00033] Indeed, in a preferred embodiment, the compositions described herein can be advantageously added to frac waste water treatment. In the fracking process, after a well has been fracked, the water is pumped back into a pit or frac tanks for storage and settlement. It is advantageous to biologically treat the water for neutralization of certain organic compounds as well as biological materials within said water. Accordingly, an appropriate amount of the composition can be administered to the waste water pit or tank, just as it was added to the production water, for treatment. After treatment, the water settles into the pit and clean water can be skimmed from the surface of the tank or pit and reused in another well.

[00034] In certain areas, depending on rules and regulations, this skimmed water may also be appropriately returned to the soil or natural bodies of water, as the composition of the invention is non-oxidizing, so it does not pollute the water. Accordingly, this allows the waste water to be easily re-utilized in the industry, or returned to nature after appropriate treatment. In certain embodiments, further downstream processing, such as through RO-DI or carbonaceous systems may also be required to remove further materials that cannot be removed by the composition alone. Therefore, an appropriate method of administration can ameliorate and treat frack waste water. Furthermore, it is apparent of the suitable uses of the compositions for effecting such treatment.

[00035] A preferred embodiment of the invention comprises a method of treatment of paraffin build-up in a well. In such embodiments, the composition is made into a highly concentrated liquid which is injected into a well either in a batch one time or continuously to eliminate plugging caused by the production of paraffin during the normal production of oil or gas. The product is injected into the well thru a low pressure pump and allowed to remain in the well for a prescribed period of time. The composition dissolves the paraffin and unplugs the wellbore and restores production. Because of the nature of the bacteria, the paraffin is removed and replaced by a biofilm. Upon removal of all food sources, the bacterial will then return to a dormant state for reactivation upon activation. The

concentration of the liquid will depend on several factors including the total depth of the well, the total volume, the amount of paraffin build-up, and the amount of additional biologic materials in the well. Appropriate concentrations can be generated by one of skill in the art.

[00036] Accordingly, the invention is formulated to control bacterial biofilms as well as to produce natural surfactants and antibiotics in situ to make the fracking process more efficient as well as vastly reducing the requirement for expensive, hazardous chemicals.

[00037] The invention is also highly effective at remediating flowback fluids and reduce biological and chemical oxygen demand to a point where they can be safely discharged to surface water or underground, compliant with NPDES / UIC regulations.

[00038] Finally, the invention can be suitable utilized to treat paraffin build-up in a frack well, through application of a concentrated liquid to eliminate the plugging of the well caused by paraffin building during the normal production of oil or gas.

[00039] The invention has a broad biocidal spectrum - including against sulphate and iron reducing bacteria, acid producing bacteria, leading to less corrosion, less reliance on toxic corrosion inhibitors as well as eliminating H2S production thus limiting thiocorrosion, sulphide stress cracking, sour gas production and plugging from FeS.

[00040] The Invention controls the bacterial biofilms associated with corrosion of tanks / piping, biofouling, plugging and bacterial attack of chemicals used in fracking processes; the Invention has broad range of tolerance to pressure, temperature, pH and monovalent / divalent salt cations; the Invention is compatible with common frack fluids.

[00041] The bacterial strains employed in the Invention have GRAS (Generally

Regarded as Safe) status with the EPA and cannot cause human disease. The probiotic bacteria, and the enzymes, antibiotics and surfactants they produce are non-toxic and non- corrosive. The bacteria produce a range of natural antimicrobial agents for biofilm control which are not used in medicines and therefore cannot drive resistance to therapeutic antibiotics.

[00042] The bacterial element of the invention is a prolific producer of antibiotics effective against a range of pathogens including S. aureus, E. coli, Helicobacter pylori (Pinchuk et al, 2001), Campylobacter (Sorokulova, Kirik and Pinchuk, 1997) and one lanthionine-containing peptide antibiotic (lantibiotic) produced, subtilin, has a broad spectrum antimicrobial activity - by forming transmembrane pores it depolarises energized bacterial cytoplasmic membranes.

[00043] The macrolide lipopeptide produced by the Invention has a powerful, broad- spectrum antimicrobial action but also is one of the most powerful biosurfactants known, reducing the surface tension of water from 72mNm-l to 27mNm-l at concentrations as low as 20μΜ. Much of the biosurfactant potency comes from the molecule being able to occupy the intermolecular space between water molecules and thus disrupting the van der Waals forces between the slight electrical dipole in individual H20 molecules.

[00044] This biosurfactant molecule is entirely natural and can aid not just the inj ection and recovery process but the drilling process too, depending on the lithographic profile. It eliminates the need for exogenous surfactant as well as dramatically decreasing the energy input to the process. [00045] Controlling and re-engineering the ecology of problem biofilms using the invention offers long term control and protection because the bacteria are not 'used up' as a biocide would be. The invention's activity against other microorganisms is threefold: it has a cation carrier effect, forms transmembrane pores in competing microorganisms and is also able to insert its lactone-bonded β-hydroxy 13-15C fatty acid chain into cell membranes thus turning unwanted bacteria initially into self-associating mixed micelles and so ultimately causing bilayer solubilisation.

[00046] It should also be noted that both batch and on-the-fly attempts at attaining environmental sterility are doomed to failure due to the recalcitrance of biofilms and the fact that tanks are rarely completely emptied - and when they are refilled the inevitable remaining bacteria thrive.

[00047] Accordingly, using the compositions for biofilm control and surfactant production can minimize environmental hazards as well as dramatically improving efficiency - thus further lowering carbon footprint. It is also extremely effective at the bioremediation of waste water and contaminated land.

[00048] In a further preferred embodiment, a kit is suitably provide comprising a predetermined amount of B. subtilis spores, wherein said spores were generated at 48°C in a medium containing elevated levels of divalent cations and pyridine-2,6-dicarboxylic acid (dipicolinic acid (DP A)) to decrease spore water content for optimum wet and dry heat resistance. The medium also contains elevated levels of a/b-type small acid-soluble spore proteins (SASP) which bind to spore DNA and protect it from damage by other agents such as UV radiation and toxic chemicals. Further included in the kit is GFAK and a 50 mM- Tris.HCL buffer at pH 7.5. Further included are instructions on how to dissolve the components into water and apply to fracking water, frack waste water, or to dissolve into a concentrate for application for dissolving and remediation of paraffin build-up. [00049] The composition can therefore effectively be used to treat bacterial build-up, including biofilm within a frack water tank, within a frack well. Further use includes treatment and remediation of frack waste water, wherein the composition can be suitably utilized to neutralize bacteria and certain organic compounds within said waste water.

Finally, the composition may be suitably utilized for remediation and removal of paraffin build-up in a frack well or in oil and gas components.

[00050] Example 1 :

[00051] A composition is generated wherein the bacteria are sporulated at 48°C in a medium containing elevated levels of divalent cations and pyridine-2,6-dicarboxylic acid (dipicolinic acid (DPA)) to decrease spore water content for optimum wet and dry heat resistance. The medium also contains elevated levels of a/b-type small acid-soluble spore proteins (SASP) which bind to spore DNA and protect it from damage by other agents such as UV radiation and toxic chemicals. The spores are thereafter combined with GFAK in a 50 mM-Tris/HCL buffer at pH 7.5 for activation and then added to frac water.

[00052] A test was performed to evaluate the efficacy of B. subtilis compositions in reducing sulphite reducing bacteria (SRBs) using the B. subtilis compositions. The tests were performed according to BSEN1276:2009, chemical disinfectants & antiseptics - quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas. Test method and requires (Phase 2, Step 1), with consideration being given to NACE TM0194.

[00053] Naturally occurring SRBs were isolated from pond sludge and utilizes as the test organism culture. 8 ml of a suspension of the SRBs were prepared in a l/500^th nutrient broth, 1 ml Bovine albumin (3.0g'l) was added with 1 ml of a suspension of B. subtilis at 10 E8 to 10 E9/ml. Care was taken so that anaerobic conditions were maintained throughout the test. [00054] Test samples were stored at 4C for 24 hours, with recoveries of SRBs being performed at 5 minutes, 60 minutes, and 24 hours. A control using an SRB suspension containing no B. subtilis was performed to compare. Tests were duplicated on two consecutive days.

[00055] Test 1

Bacillus subtilis inoculation level 0 min = 7.2x10 E8/ml

SRBs recovered/ml

[00056] Test 2

Bacillus subtilis inoculation levels 0 min = 6.4x10 E8/ml

SRBs recovered/ml

References

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711-45.

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Bacteriol., August, 70(2), pp. 192-200.

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Blood. J. Hygiene, Volume 38, pp. 732-749.

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Nov, 147(11), pp. 3121-3126.

15. ) Nakano, M. N. & Zuber, P., 1998. Anerobic Growth of a "Strict Aerobe" (Bacillus subtilis). Ann Rev Microbiol, October, Volume 52, pp. 165-190. Osipova, I., Sorokulova, I. B., Tereshkina, N. V. & Grigoreva, L. V., 1998. Safety of bacteria of the genus Bacillus, forming the base of some probiotics. Zh Mikrobiol Epidemiol Immunobiol, Nov-Dec, Volume 6, pp. 68-70.

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Pathogens. J Travel Med, December, 4(4), pp. 167-70.

Claims

Claims

1. A composition suitable for addition to frack water comprising: a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl in 50mM-Tris / HCl buffer, pH 7.5 (GFAK); wherein on germination the bacteria produce prodigious amounts of the natural biosurfactant surfactin, biocidal concentrations of subtilin and lanthionine-containing peptide antibiotics which act to competitively exclude unwanted bacteria; in addition they produce a-amylase enzymes which detach biofilm exopolysaccharide components; when added to fracking fluids they increase efficiency, control biofilm formation and reduce or remove the need to add environmentally-damaging chemicals to frack fluids.

2. The composition of claim 1 wherein administration of the composition controls biofilms that hamper secondary oil and gas recovery in fracking operations as well as producing biosurfactants to increase the efficiency of fracking processes.

3. The composition of claim 1 wherein administration of the composition removes the need for synthetic chemical biocides in fracking operations and reduces or removes the need for the addition of chemical surfactants.

4. The composition of claim 1 wherein administration of the composition remediates waste waters reducing biological and chemical oxygen demand to levels where the wastes are safe to discharge.

5. The composition of claim 1 wherein the composition generates surfactin.

6. The composition of claim 1 wherein the composition generates subtilin.

7. Use of the composition of claim 1 for producing biosurfactants to increase the efficiency of fracking processes.

8. Use of the composition of claim 1 for reducing or removing the need for addition of chemical surfactants in the fracking process.

9. Use of the composition of claim 1 in reducing the biological and chemical oxygen demands of fracking waste water, wherein said reduction in biological and chemical demands are reduced such that the waste water is safe for discharge.

10. A method of introducing a benign biofilm to a surface comprising:

a. Administering a composition comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KC1 in 50mM-Tris / HC1 buffer, pH 7.5 (GFAK) to fracking water;

b. Wherein on germination, the bacteria produce prodigious amounts of the natural biosurfactant surfactin, biocidal concentrations of subtilin and lanthionine-containing peptide antibiotics.

11. The method of claim 10, wherein the surfactin, subtilin, and lanthionine- containing peptide antibiotics act to competitively exclude unwanted bacteria.

12. The method of claim 11, wherein the bacteria further produce a-amylase enzymes which detach biofilm exopolysaccharide components; and, when added to fracking fluids they increase efficiency, control biofilm formation and reduce or remove the need to add environmentally-damaging chemicals to frack fluids

A method of increasing fracking efficiency comprising:

administering a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β- fructose, 30mM L-asparagine and 6 mM KC1 in 50mM-Tris / HC1 buffer, pH 7.5 (GFAK) to fracking water; and

germinating said Bacillus spores under conditions sufficient for the bacteria to produce surfactin, subtilin, and lanthionine-containing peptide antibiotics.

14. The method of claim 13 wherein said blend of Bacillus species is selected from the group consisting of 1A5, 1A28, 1A84, 1A122, 1A698, 1A699, 1A751, and 1A752, and combinations thereof, in a concentration of about lxlO⁸ CFU ml^"1

15. A method of biofilm control and remediation of waste frack fluids comprising: a. administering to said waste frack fluids a composition comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KC1 in 50mM-Tris / HC1 buffer, pH 7.5 (GFAK);

b. germinating said Bacillus spores under conditions sufficient for the bacteria to produce surfactin, subtilin, and lanthionine-containing peptide antibiotics.

16. A method for remediating frack waste water comprising:

a. administering to said waste frack fluids a composition comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KC1 in 50mM-Tris / HC1 buffer, pH 7.5 (GFAK); b. germinating said Bacillus spores under conditions sufficient for the bacteria to produce surfactin, subtilin, and lanthionine-containing peptide antibiotics.

17. A method for paraffin removal from a well comprising:

a. administering to said well a concentrated fluid comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl in 50mM-Tris / HCl buffer, pH 7.5 (GFAK);

b. germinating said Bacillus spores under conditions sufficient for the bacteria to produce surfactin, subtilin, and lanthionine-containing peptide antibiotics.

18. A composition having a broad biocidal spectrum comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl in 50mM-Tris / HCl buffer, pH 7.5 (GFAK); wherein said composition's broad biocidal spectrum is effective against sulphate and iron reducing bacteria, acid producing bacteria, thereby leading to less corrosion, less reliance on toxic corrosion inhibitors as well as eliminating ¾S production thus limiting thiocorrosion, sulphide stress cracking, sour gas production and plugging from FeS.

19. A composition having a broad biocidal spectrum comprising a blend of Bacillus species, 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl in 50mM-Tris / HCl buffer, pH 7.5 (GFAK), which is suitable for being dissolved into an aqueous medium for germinating said species.

20. Use of the composition of claim 19 in generating benign biofilms.

21. Use of the composition of claim 19 in remediating frack waste water.

22. Use of the composition of claim 19 in reducing the presence of paraffin on a surface.

23. A kit for treatment of frack water comprising: a. a predetermined amount of B. subtilis spores, wherein said spores were generated at 48°C in a medium containing elevated levels of divalent cations and pyridine-2,6-dicarboxylic acid (dipicolinic acid (DP A)) to decrease spore water content for optimum wet and dry heat resistance; and wherein the medium also contains elevated levels of a/b-type small acid-soluble spore proteins (SASP) which bind to spore DNA and protect it from damage by other agents such as UV radiation and toxic chemicals;

b. 5.6mM β-D-glucose, 5.6mM β-fructose, 30mM L-asparagine and 6 mM KCl (GFAK);

c. 50 mM-Tris HCL buffer at pH 7.5; and

d. instructions on how to dissolve the components into water and apply to

fracking water, frack waste water, or to dissolve into a concentrate for application for dissolving and remediation of paraffin build-up.