Patent Application
20170260467
The present invention relates generally to antimicrobials and, more specifically, to methods of controlling microbial growth and proliferation.
Microorganisms are highly adaptable to surrounding environments, which allows cultures to colonize nearly any environment. Some microorganism cultures are resistant to very recalcitrant pollutants including, for example, polychlorinated biphenyls, heavy metals, and hydrocarbon fuels.
Bacteria have been isolated from fuels, fuel storage tanks, pipelines, aircraft wing tanks, and offshore oil platforms, in which the bacteria may cause problems such as tank corrosion, fuel pump failures, filter plugging, injector fouling, topcoat peeling, engine damage, and deterioration of fuel chemical properties and quality. Extensive microbial growth and biofilm formation within the fuel, fuel tanks, or fuel lines may also lead to costly and disruptive damage to fuel systems. These besides have the ability to metabolize hydrocarbons and thrive in the environments containing toxic compounds (i.e., aromatic hydrocarbons), low nutrient availability (metal ions, phosphorus, etc.), and low water amounts.
Normally, bacteria metabolize alkanes via oxidation. However, the genome of bacteria adapted to grow in in jet-fuel systems and petroleum oil field (such as P. aeruginosa) encodes two membranes bound alkane hydroxylases (alkB1 and alkB2), essential electron transfer proteins, ruberdoxins (RubA1, RubA2), and FAD dependent NAD(P)H2 ruberdoxin reductases, which oxidize a terminal methyl group of the alkanes into a primary alcohol group via alkane hydroxylases aided with electron transfer proteins. The primary alcohol group is oxidized to an aldehyde and a fatty acid and followed by β-oxidation to generate acetyl-CoA, the entry molecule for the citric acid cycle.
The role of membrane proteins and cell membrane is crucial in regulating cell homeostasis. One class of membrane proteins, encoded by the opr genes, includes substrate specific porins that transport molecules from the extracellular environment into the cell. Two such porins, OprF and OprG, are involved in the transport of aromatic hydrocarbons and other hydrophobic small molecules into the cells. Fuel contains aromatic and cyclic hydrocarbons, which are toxic to the cell. Also, fuel can capture heavy metals and other molecules during transport and storage, which may also affect bacteria. It has been proposed that membrane proton antiporter-pumps or efflux pumps of the resistance-nodulation-division (“RND”) family function in the extrusion of toxic compounds including antimicrobials, organic solvents, and heavy metals.
Despite the current understanding of bacterial growth in fuels, there remains a need for methods of controlling and/or preventing such bacterial growth and other microbes that are responsible for biodeterioration of the fuel.
The present invention overcomes the foregoing problems and other shortcomings, drawbacks, and challenges of controlling or preventing microbial biodeterioration of fuel. While the invention will be described in connection with certain embodiments, it will be understood that the invention is not limited to these embodiments. To the contrary, this invention includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the present invention.
According to one embodiment of the present invention, a method of preventing biodeterioration of a fuel by reducing a microbial growth in the fuel includes administering an antimicrobial peptide to a fuel phase of the fuel, an aqueous phase of the fuel, or both. The antimicrobial peptide is configured to disrupt cellular membranes of the microbes compromising the growth and includes antimicrobial peptides having a β-sheet conformation, an α-helix conformation, or a combination thereof.
In accordance with another embodiment of the present invention, a method of preventing biodeterioration of a fuel by reducing a microbial growth in the fuel includes administering an efflux pump inhibitor to a fuel phase of the fuel, an aqueous phase of the fuel, or both. The efflux pump inhibitor is configured to block an efflux transport of toxins by efflux pumps or porins from microbes comprising the growth. The efflux pump inhibitor is selected from a group consisting of peptidomimetic, a c-capped dipeptide, an antibody, a nanobody, and nucleic acid, an aptamer, a peptide with second, tertiary, or quaternary structure configured to block efflux pumps or porins, and a small chemical molecule configured to block efflux pumps or porins.
Yet another embodiment of the present invention is directed to an antimicrobial fuel comprising a fuel phase and an aqueous phase at least partially separated from the fuel phase. An effective concentration of an antimicrobial peptide is in the fuel phase, the aqueous phase, or both, and is configured to disrupt a cellular membrane of microbes within the fuel.
Still another embodiment of the present invention is directed to an antimicrobial fuel comprising a fuel phase and an aqueous phase at least partially separated from the fuel phase. An effective concentration of an efflux pump inhibitor is in the fuel phase, the aqueous phase, or both, and is configured to block an efflux transport of toxins by at least one efflux pump of microbes in the fuel.
According to another embodiment of the present invention, a fuel treatment solution includes a lyophilized antimicrobial peptide, a lyophilized efflux pump inhibitor, or both dissolved in an amphipathic solvent.
According to one aspect of the present invention, the fuel treatment solution may be administered to a fuel phase of a fuel. The fuel treatment solution migrates from the fuel phase to an aqueous phase and inhibits microbial growth.
Additional objects, advantages, and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be leaned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present invention and, together with a general description of the invention given above, and the detailed description of the embodiments given below, serve to explain the principles of the present invention.
It should be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features illustrative of the basic principles of the invention. The specific design features of the sequence of operations as disclosed herein, including, for example, specific dimensions, orientations, locations, and shapes of various illustrated components, will be determined in part by the particular intended application and use environment. Certain features of the illustrated embodiments have been enlarged or distorted relative to others to facilitate visualization and clear understanding. In particular, thin features may be thickened, for example, for clarity or illustration.
Turning now to the figures, and in particular to
With volume of the fuel, an effective concentration of antimicrobial peptide is determined (Block 14). The effective concentration depends, in part, on a selected antimicrobial peptide, which generally includes peptides having a β-sheet conformation, an α-helix conformation, or both.
The effective concentration may also depend, in part, on an identity of the microbial culture, which may include environmental, fuel-degrading bacteria (for example, Pseudomonas, Bacillus, Achromobacter, Marinobacter, Rhodovumlum, Dietzia, Halobacillus, Acinetobacter, Alcaligenes, Nocardioides, Rhodococcus, Methylobacterium, Loktanella, Escherichia, and Staphylococcus), fungi (for example, Yarrowia, Hormoconis, and Cladosporium), or combinations thereof. In that regard, and if desired, an identity of the microbial culture may be determined (Block 16) and may include a cell count or density, for example, ranging 1 cell per mL fuel to 1×109 cells per mL fuel, although these cell densities are not limiting. Effective concentrations may range from about 1 μg/mL to about 100 μg/mL (or about 1 ppm to about 100 ppm), but is generally considered to be a minimum concentration at which the microbial culture growth decreases by 85% to 100%.
The effective concentration of the antimicrobial peptide is administered to the fuel phase, the aqueous phase, or both phases of the fuel (Block 18). After a desired time, for example, ranging from 24 hours to several days (four or more days), control of microbial growth is determined (Block 20). If microbial densities are less than 0.2 OD or 1×106 cell/mL, then microbial growth is controlled (“Yes” branch of Decision Block 20) and the process ends. However, if microbial growth is greater than 0.2 OD or 1×106 cell/mL, then microbial growth is not controlled (“No” branch of Decision Block 20) and the process returns to again determine the volume of the fuel (Block 12).
Alternatively, and as shown in
Antimicrobial peptides are peptides produced and utilized by animals to protect again microorganisms. Generally, antimicrobial peptides are non-discriminatory against bacteria, fungi, and viruses by interacting directly with cell membranes rather than with specific proteins within the membranes. In that regard, the antimicrobial peptides may permeate and destabilize the cell membrane, leading to cellular death. Two examples of highly active, small antimicrobial peptides include Protegrin-1 (PG-1) and Magainin-2. PG-1 is an 18 amino acid cysteine-rich β-sheet peptide while Magainin-2 is 23-residue peptide with an α-helical conformation. Each of these peptides effectively perforates cellular membranes by agglomerating into forming pores across the membrane, which lead to cell lysis.
Turning now to
With volume of the fuel, an effective concentration of efflux pump inhibitor is determined (Block 34). The effective concentration depends, in part, on a selected efflux pump inhibitor, which, for example, may include one or more of c-capped dipeptides, Phe-Arg-β-napththylamide, and MC-207,100.
The effective concentration may also depend, in part, on an identity of the microbial culture, which may include environmental, fuel degrading bacteria (for example, Pseudomonas, Bacillus, Achromobacter, Marinobacter, Rhodovulum, Dietzia, Halobacillus, Acinetobacter, Alcaligenes, Nocardioides, Rhodococcus, Methylobacterium, Loktanella, Escherichia, and Staphylococcus) or combinations thereof. In that regard, and if desired, an identity of the microbial culture may be determined (Block 36) and may include a cell count or density, for example, ranging 1 cell per mL fuel to 1×109 cells per mL fuel, although these cell densities are not limiting. Effective concentrations may range from about 20 μg/mL to about 80 μg/mL (or about 20 ppm to about 80 ppm), but is generally considered to be a minimum concentration at which the microbial culture growth decreases by 85% to 100%.
The effective concentration of the efflux pump inhibitor is administered to the fuel phase, the aqueous phase, or both phases of the fuel (Block 38). After a desired time, for example, ranging from 24 hours to several days (four or more days), control of microbial growth is determined (Block 40). If microbial densities are less than 0.2 OD or 1×106 cell/mL, then microbial growth is controlled (“Yes” branch of Decision Block 40) and the process ends. However, if microbial growth is greater than 0.2 OD or 1×106 cell/mL, then microbial growth is not controlled (“No” branch of Decision Block 40) and the process returns to again determiner the volume of the fuel (Block 32).
Efflux pumps inhibitors may include peptidomimetics, c-capped dipeptides, dipeptide compounds, Phe-Arg-β-napthylamide and analog structures, diamine-containing peptides and analogs, compounds that competitively bind to the substrate binding sites of resistance nodulation division (“RND”) family of efflux pumps, compounds that competitively bind to the substrate binding sites of major facilitator superfamily (“MFS”) of efflux pumps, compounds that competitively bind to the substrate binding sites of ATP-binding cassette (“ABC”) superfamily of efflux pumps, allosteric inhibitors of efflux pumps, efflux pump inhibitors (such as, pyridopyrimidines, arylpiperazines, and arylpiperidines), antibodies or nanobodies raise to recognize epitopes in the efflux pumps or porins and that block efflux pump activity by binding to the efflux pump, nucleic acids, aptamers, small chemical molecules having structures configured to recognize, interact, and block efflux pumps or porins, and peptides having secondary, tertiary, or quaternary structure that is configured to bind and block efflux pumps or porins within the cellular membranes of the microbes. With the efflux pumps blocked, toxins from the fuel accumulate within the cytoplasm of the microbes and prevent microbial growth.
Alternatively, and as shown in
Efflux pumps inhibitors are peptidomimetics, c-capped dipeptides, small peptides, antibodies, nucleic acids, aptamers, small molecules, and chemicals that are configured to bind and block efflux pumps in the cellular membranes of microbes. Once blocked, the efflux pumps are prevented from exporting accumulated toxic compounds in fuel from inside the microbe, leading to growth inhibition.
With reference now to
Accordingly, and as provided in Block 64, the treatment solution may be administrated to the volume of fuel.
The following examples illustrate particular properties and advantages of some of the embodiments of the present invention. Furthermore, these are examples of reduction to practice of the present invention and confirmation that the principles described in the present invention are therefore valid but should not be construed as in any way limiting the scope of the invention. Thereafter, for example, 24 hours to several days (four or more days) microbial growth is determined as described previously. If the microbial growth is controlled (“Yes” branch of Decision Block 66), then the process ends; however, if microbial growth remains uncontrolled (“No” branch of Decision Block 66) then the process returns to further administer the treatment solution to the volume of fuel (Block 64).
Protegrin-1 and Magainin-2 antimicrobial peptides were added individually to the fuel phase and the aqueous (minimal media M9, Bushnell-Haas, or water) phase of 1:1 fuel-growth media mixtures containing environmental bacteria (E. coli, Bacillus, and Pseudomonas) at concentrations ranging from 1 to 1×109 cells/mL. Magainin 1 and 2 were obtained from Sigma-Aldrich (St. Louis, Mo.). Protegrin-1 was obtained from AnaSpec (Fremont, Calif.) or produced from a transgenic construct containing a fusion between green fluorescent protein (“GFP”) and the Protegrin-1 coding gene. The GFP-Protegrin fusion was purified by affinity chromatography and Protegrin cleaved from the fusion for use, as pure, or as a fusion in the bioassays.
The antimicrobial peptides were added at the following concentrations: 0 μg/mL, 1μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL, and 125 μg/mL in the presence and absence of fuel. Experiments using minimal media with bacteria in the presence of fuel were designed to measure the effect of fuel in combination with the antimicrobial peptide control. Control experiments contained glycerol instead of fuel as the energy source.
Addition of the antimicrobial peptides directly to the fuel phase lowered the amount of peptide required to achieve complete growth inhibition by at least two-fold. Protegrin-1 showed activity that prevented microbial growth at concentrations less than or equal to about 1 μg/mL.
The antimicrobial effect of the peptides was measured every 24 hours for four days after inoculation by measuring growth through absorbance readings (OD600), DNA quantitation through qPCR, and colony counting techniques.
The addition of antimicrobial peptides of the type Protegrin-1 and Magainin-2 to fuel (aqueous and fuel phase) partitioned into the aqueous phase and inhibited bacteria growth.
When the antimicrobial peptide Protegrin-1 was used in the presence of fuel, the concentration required to completely inhibit growth was reduced from 5 μg/mL in E. coli and Pseudomonas to less than or equal to 1 μg/mL (
C-capped dipeptide efflux pump blocker, Phe-Arg β-naphthylamide dihydrochloride (MC-207,110) (Sigma Aldrich) was added to the fuel phase and the aqueous (minimal media M9, Bushnell-Haas, or water) phase of 1:1 fuel-minimal media mixtures containing environmental bacteria (Pseudomonas, Acinetobacter, Marinobacter, and Dietzia) at concentrations ranging from 1 to 1×109 cells/mL. Phe-Arg β-naphthylamide dihydrochloride was added to the fuel at concentrations of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL. Control experiments were performed by adding 0 μg/mL to 120 μg/mL of Phe-Arg β-naphthylamide to minimal media containing bacteria and glycerol as the energy source, but not fuel.
Partial bacterial growth inhibition was observed at 20 μg/mL and complete growth inhibition was achieved at 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL of c-capped dipeptide, as shown in
The effective concentration to produce complete growth inhibition ranged from 20 μg/mL to 80 μg/mL and was dependent on the bacterial level and the length of the incubation used. Complete growth inhibition for up to 17 days was observed at concentrations greater than about 80 μg/mL (
EXAMPLE 3
Treatment solutions were prepared, as described above, with 25 mg/mL efflux pump inhibitor in various solvents, including absolute ethanol, DiEGME, and water. The treatment solutions were administrated to jet fuel at a final concentration in fuel of 0 μg/mL, 40 μg/mL, and 80 μg/mL. FIG. 12 illustrates results of the 80 μg/mL treatment on initial measured microbial growth in the aqueous phase as well as microbial growth after one, two, and three days. Treatment of the jet fuel significantly decreased microbial growth in the aqueous phase.
While the present invention has been illustrated by a description of one or more embodiments thereof and while these embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and method, and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.
This application claims the benefit of and priority to prior filed co-pending Non-Provisional Application Ser. No. 14/195,151, filed Mar. 3, 2014, which claims the benefit of and priority to prior filed, now expired, Provisional Application Ser. No. 61/829,593, filed May 31, 2013. The disclosure of each application is expressly incorporated herein by reference, in its entirety.
The invention described herein may be manufactured and used by or for the Government of the United States for all governmental purposes without the payment of any royalty.
| Number | Date | Country | |
|---|---|---|---|
| 61829593 | May 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14195151 | Mar 2014 | US |
| Child | 15609988 | US |
