# Patent

## Title
Altering the interface of hydrocarbon-coated surfaces

## Abstract

Methods and compositions are provided wherein microorganisms are used to alter the interface of hydrocarbons and hydrocarbon-coated surfaces to increase oil recovery, for improved bioremediation and/or to benefit pipeline maintenance.

## Background
This application is a Continuation in Part claiming priority to U.S. application Ser. No. 12/784,518 filed May 21, 2010, now pending, which in turn claims priority to U.S. Provisional Applications 61/180,529 and 61/180,445, each filed on May 22, 2009.


FIELD OF INVENTION
This invention relates to the field of environmental microbiology and modification of heavy crude oil properties using microorganisms. More specifically, microorganisms are used to alter the interface between hydrocarbons and a surface to increase oil recovery from hydrocarbon coated surfaces.
BACKGROUND OF THE INVENTION
Hydrocarbons in the form of petroleum deposits and oil reservoirs are distributed worldwide. These oil reservoirs are measured in the hundreds of billions of recoverable barrels. Because heavy crude oil has a relatively high viscosity and may adhere to surfaces, it is essentially immobile and cannot be easily recovered by conventional primary and secondary means.
Microbial Enhanced Oil Recovery (MEOR) is a methodology for increasing oil recovery by the action of microorganisms (Brown, L. R., Vadie, A. A, Stephen, O. J. SPE 59306, SPE/DOE Improved Oil Recovery Symposium, Oklahoma, Apr. 3-5, 2000). MEOR research and development is an ongoing effort directed at discovering techniques to use microorganisms to benefit oil recovery (Sunde. E., Beeder, J., Nilsen, R. K. Torsvik, T., SPE 24204, SPE/DOE 8th Symposium on enhanced Oil Recovery, Tulsa, Okla., USA, Apr. 22-24, 1992). An effective MEOR treatment for crude oil desorption and mobilization could utilize microbially derived surface active agents (McInerney, M. J., et al., Development of microorganisms with improved transport and biosurfactant activity for enhanced oil recovery. DE-FE-02NT15321. DOE, 2003). Few have been identified that have been shown to alter the surface interaction between hydrocarbons and rocks, soil, brine, sand or clay to which the hydrocarbons are adhered.
Use of surface active agents or surfactants to increase solubility of oil through reduction in surface and interfacial tensions is another technique for increasing oil recovery. A wide variety of surfactants identified thus far are able to significantly reduce surface and interfacial tensions at the oil/water and air/water interfaces. Because surfactants partition at oil/water interfaces, they are capable of increasing the solubility and bioavailability of hydrocarbons (Desai, J. D. and I. M. Banat. Microbial production of surfactants and their commercial potential. Microbiol. Mol. Biol. Rev., 47-64, 1997 and Banat, I. M. Bioresource Technol. 51: 1-12, 1995 and Kukukina, M. S., et al. Environment International. 31: 155-161, 2005 and Mulligan, C., Environmental Pollution. 133: 183-198, 2005). Doong and Lei (J. Hazardous Materials. B96: 15-27, 2003), for example, found that the addition of surfactants to soil environments contaminated with polyaromatic hydrocarbons increased the mineralization rate of some hydrocarbons (Doong, R and W. Lei, supra). Such surfactants are expensive and may pose environmental or other equipment issues.
Biosurfactants, (biologically produced surfactants), have helped to substantially increase oil recovery from sandstone deposits by increasing solubility and decreasing viscosity of the oil (Mulligan, C., supra). Depending on the application, biosurfactants may be preferred since they are generally more biodegradable and less toxic than synthetically produced surfactants, and are effective under a broad range of oil and reservoir conditions. Examples of biosurfactants include glycolipids, lipopeptides and lipoproteins, fatty acids and phospholipids, polymeric compounds, and particulate biosurfactants (Desai, J. D. supra). However, further characterization of production and use of biosurfactants is needed. Further, there is a need to identify microorganisms that are able to produce these biosurfactants under reservoir conditions or other relevant environmental conditions.
Certain microorganisms have been described as having properties that may benefit MEOR processes. Certain Shewanella species have been disclosed as useful for remediation of metal contamination (U.S. Pat. No. 6,923,914B2), iron containing mixed waste (U.S. Pat. No. 6,719,902B1), manganese contamination (U.S. Pat. No. 6,350,605B1), and other pollutants with the aid of butane (U.S. Pat. No. 6,245,235B1). In EP1189843, certain Shewanella species were described as being useful for bioremediation of petroleum contaminants aerobically. In addition, Shewanella supplemented with butane was used for reduction of fouling in injection and recovery wells under aerobic conditions (U.S. Pat. No. 6,244,346B1). Other Shewanella species have been described as having the ability to produce biofilms (D. Bagge, et al., Appl. Environ. Microbiol. 67, 2319-2325. 2001); to sequester gases, in particular CO2, in underground geological formations and prevent their release into the atmosphere (see US20060216811A1); and to enhance oil recovery (commonly owned and co-pending US 2009-0260803 A1). The activity reported by these microorganisms is related to the degradation and transformation of hydrocarbons and other pollutants and not related to altering the interfacial boundaries between hydrocarbons and the surfaces to which they are bound.
The problem to be solved therefore, relates to the identification of microorganisms that: 1) have the ability to alter the interface between hydrocarbons and rock or other surfaces subject to coating by oil; 2) can be inoculated under suitable conditions which effect these alterations in surface properties; and 3) can be used in a cost-efficient way, to improve oil recovery, and benefit bioremediation.
SUMMARY OF THE INVENTION
The methods described herein solve the stated problem above, by identifying microorganisms that have the ability to alter the interface between hydrocarbons and the surfaces which they coat in order to improve oil recovery, and benefit bioremediation. The alterations result in substantial liberation of oil from hydrocarbon-coated surfaces. In one aspect the microorganisms are Shewanella species that have the ability to affect the wettability of the surfaces through microbial action. In addition, a new isolate of Shewanella sp. has been identified.
Accordingly invention provides a method for altering the wettability of a hydrocarbon coated surface comprising:

    
    
        a) providing a hydrocarbon-coated surface;
        b) providing a medium selected from the group consisting of:
        
            i) a cell-containing medium comprising one or more Shewanella sp.; and
            ii) a conditioned medium which is substantially cell free and which has been in contact with one or more Shewanella sp.;
            wherein the Shewanella sp. comprises a 16S rDNA comprising SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:28; and
        
        
        c) contacting said hydrocarbon-coated surface with the medium of b) wherein the medium alters the wettability of said hydrocarbon-coated surface.
    
    


In another aspect the invention provides a method of oil recovery comprising:

    
    
        a) providing an oil reservoir;
        b) injecting the oil reservoir with the medium of claim 1; and
        c) recovering oil from the oil reservoir;

wherein the medium enhances oil recovery.

    
    


In another aspect the invention provides a method of treating an environmental site comprising:

    
    
        a) providing an environmental site comprising hydrocarbon-coated surfaces;
        b) contacting the environmental site with the medium of claim 1 wherein the hydrocarbon is released from the site;
        c) collecting water of the medium and the released hydrocarbon of (b);
        d) separating the hydrocarbon and water; and
        e) making medium of claim 1 using the water of (d) for use in (b).
    
    


In another aspect the invention provides a composition comprising:

    
    
        a) at least one Shewanella sp. comprising a 16S rDNA comprising SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:28; and
        b) an electron acceptor selected from the group consisting of nitrate, fumarate, ferric ion, manganese (MnIV) ion and mixtures thereof.
    
    





BRIEF DESCRIPTION OF FIGURES AND SEQUENCES
The invention can be more fully understood from the following detailed description, the Figures, and the accompanying sequence descriptions, which form a part of this application.
FIG. 1. Depicts oil release over time with early and late stage growth of strain LH4:18.
FIG. 2. Depicts oil release over time comparing aerobic growth versus anaerobic growth.
FIG. 3. Depicts oil release and MPNs of strain LH4:18 in the presence of different electron acceptors.
FIG. 4. Depicts oil release over time with strain LH4:18 grown in the presence or absence of glucose.
FIG. 5. Depicts oil release over time with strain LH4:18 grown in the presence of different media and supplements. Where indicated, supplements were 1% peptone, 1.6 mM MgSO4, 20 mM KCl, 20 mM NH4Cl, and 20 mM tris base.
FIG. 6. Depicts oil release over time with strain LH4:18 grown in simulated injection brine supplemented with peptone or yeast extract.
FIG. 7. Depicts oil release over time with strain LH4:18 culture and supernatant alone.
FIG. 8. Depicts oil release over time with strain LH4:18 supernatant (centrifuged compared with centrifuged and filtered) and strain LH4:18 cell pellet resuspended in fresh medium.
FIG. 9. Depicts oil release over time with strain LH4:18 in the presence and absence of Pseudomonas stutzeri, species LH4:15.
FIG. 10. Depicts oil release over time with LH4:18 and other Shewanella species (strains EH60:12, EH60:2, and EH60:10).
FIG. 11. Depicts oil release over time with LH4:18 and other Shewanella species purchased through DSMZ (Deutsche Sammlung von Mikrorganismen and Zellkulturen, German Collection of Microorganisms and Cell Cultures).
FIG. 12. Pictograph of contact angle comparisons between untreated oil coated sand (A) and strain LH4:18 treated oil coated sand (B).
FIG. 13. Depicts residual oil versus position along sandpack tubes comparing strain LH4:18 treated and untreated sandpacks.
FIG. 14. Shewanella species alignment for signature sequence region 3.
FIG. 15. Shewanella species alignment for signature sequence region 6.
FIG. 16. Riboprint® batch report, 052009, used for comparisons of Shewanella sp. L3:3 Riboprint® #212-824-S-4 to other Shewanella Riboprint® s in the Qualicon and DuPont Environmental Sciences Riboprint® Databases.
FIGS. 17A and B. Pictograph of contact angle comparisons between untreated oil coated sand (A) and L3:3 treated oil coated sand (B).
FIG. 18 shows dominant and degenerate signature sequences for Shewanella species in rDNA variable regions 2 (A), 5 (B), and 8 (C). The variable positions are underlined. Alternative nucleotides for each variable position designation are given in the legend. Shewanella oneidensis MR-1 is representative of Shewanella having the dominant signature sequences.
FIG. 19 is a graph of oil release over time in a LOOS test using strain MPHPW-1 as compared to a medium alone control.
FIG. 20 is a graph of oil release over time in a LOOS test using strain MPHPW-1 grown with different nutrients added to growth medium.
FIG. 21 is a diagram of the sandpack apparatus used to demonstrate oil release.
FIG. 22 is a graph of water saturation before and after treatment with strain MPHPW-1.
FIG. 23 is a graph of oil release over time in a LOOS test using strain MPHPW-1 sandpack inoculum compared to a medium alone control.
FIG. 24 is Guide Tree, resembling a phylogenetic tree, showing the genus Shewanella type strains, Shewanella algae strain BrY, and strain MPHPW-1. The tree was prepared based on a sequence distance method and utilizes Neighbor Joining algorithm of Saitou and Nei from 1987 (infra).
FIG. 25 shows a Riboprint® analysis of strain MPHPW-1 and related Shewanella strains.



The following DNA sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for patent applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NO:1 is oligonucleotide primer 1492R.
SEQ ID NO:2 is oligonucleotide primer 8F.
SEQ ID NO:3 is 16S rDNA from Shewanella sp. L3:3
SEQ ID NO:4 is 16S rDNA from CP000681 Shewanella putrefaciens CN-32.
SEQ ID NO:5 is 16S rDNA from Shewanella putrefaciens LH4:18.
SEQ ID NO:6 is 16S rDNA FJ210800 from Shewanella algae. 
SEQ ID NO:7 is 16S rDNA EU563337.1 from Shewanella sp. C13-M.
SEQ ID NO:8 is 16S rDNA EU563345.1 from Shewanella sp. C31.
SEQ ID NO:9 is 16S rDNA DQ164801.1 from Shewanella sp. L-10.
SEQ ID NO:10 is 16S rDNA FM210033.2 from Shewanella chilikensis JC5T.
SEQ ID NO:11 is 16S rDNA EU721813 from Shewanella uncultured clone D004024H07.
SEQ ID NO:12 is 16S rDNA EU563338.1 from Shewanella sp. C16-M.
SEQ ID NO:13 is the DNA sequence corresponding to prokaryote 16S rRNA variable region 3 that is signature to Shewanella sp. L3:3 and related strains.
SEQ ID NO:14 is the DNA sequence corresponding to prokaryote 16S rRNA variable region 6 that is signature to Shewanella sp. L3:3 and related strains.
SEQ ID NO:15 is a partial sequence of the 16S rDNA of Shewanella sp. strain EH60:12.
SEQ ID NO:16 is a partial sequence of the 16S rDNA of Shewanella sp. strain EH60:10.
SEQ ID NO:17 is a partial sequence of the 16S rDNA of Shewanella sp. strain EH60:2.
SEQ ID NO:18 is the Shewanella dominant signature sequence for the 16S rDNA variable region 2.
SEQ ID NO:19 is the Shewanella degenerate signature sequence for the 16S rDNA variable region 2.
SEQ ID NO:20 is the Shewanella dominant signature sequence for the 16S rDNA variable region 5.
SEQ ID NO:21 is the Shewanella degenerate signature sequence for the 16S rDNA variable region 5.
SEQ ID NO:22 is the Shewanella dominant signature sequence for the 16S rDNA variable region 8.
SEQ ID NO:23 is the Shewanella degenerate signature sequence for the 16S rDNA variable region 8.
SEQ ID NO:24 is 16S rDNA from Shewanella algae MPHPW-1
SEQ ID NO:25 is the Shewanella degenerate signature sequence for the 16S rDNA variable region 2 adjusted at position 23 to include the MPHPW-1 16S rDNA sequence.
SEQ ID NO:26 is 16S rDNA from strain IBI-6P, which is SEQ ID 3 from US20100044304.
SEQ ID NO:27 is 16S rDNA from X81621 Shewanella algae BrY.
SEQ ID NO:28 is the Shewanella degenerate signature sequence for the 16S rDNA variable region 2 containing the MPHPW-1 distinguishing C at position 23.
SEQ ID NO:29 is the DNA sequence corresponding to prokaryote 16S rRNA variable region 3 that is signature to Shewanella sp. MPHPW-1.
SEQ ID NO:30 is Genbank Accession No. X82131 Shewanella benthica 16S rRNA gene (ATCC 43992) type strain.
SEQ ID NO:31 is Genbank Accession No. X82132 Shewanella hanedai 16S rRNA gene (CIP 103207T) type strain.
SEQ ID NO:32 is Genbank Accession No. X81623 Shewanella putrefaciens 16S rRNA gene type strain.
SEQ ID NO:33 is Genbank Accession No. EF178282 Shewanella haliotis strain DW01 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:34 is Genbank Accession No. AJ311964 Shewanella denitrificans partial 16S rRNA gene, strain OS-217 type strain.
SEQ ID NO:35 is Genbank Accession No. EU143361 Shewanella sp. J83 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:36 is Genbank Accession No. AF420312 Shewanella fidelia strain KMM3582T 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:37 is Genbank Accession No. EU290154 Shewanella marinus strain C4 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:38 is Genbank Accession No. U85903 SFU85903 Shewanella frigidimarina ACAM 591T 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:39 is Genbank Accession No. U85907 SGU85907 Shewanella gelidimarina ACAM456 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:40 is Genbank Accession No. AJ300834 Shewanella livingstonis 16S rRNA gene, strain LMG19866T type strain.
SEQ ID NO:41 is Genbank Accession No. AM980877 Shewanella vesiculosa partial 16S rRNA gene, type strain M7T type strain.
SEQ ID NO:42 is Genbank Accession No. D21225 VIB16SRRF Shewanella violacea gene for 16S rRNA, partial sequence type strain.
SEQ ID NO:43 is Genbank Accession No. FJ589031 Shewanella sp. S4 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:44 is Genbank Accession No. AF005248 Shewanella amazonensis 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:45 is Genbank Accession No. AF005249 Shewanella algae strain ATCC 51192 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:46 is Genbank Accession No. AF005251 Shewanella oneidensis MR-1 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:47 is Genbank Accession No. AB081757 Shewanella marinintestina gene for 16S rRNA, partial sequence type strain.
SEQ ID NO:48 is Genbank Accession No. AB081760 Shewanella schlegeliana gene for 16S rRNA, partial sequence type strain.
SEQ ID NO:49 is Genbank Accession No. AB081762 Shewanella sairae gene for 16S rRNA, partial sequence type strain.
SEQ ID NO:50 is Genbank Accession No. FM203122 Shewanella sp. JC15 partial 16S rRNA gene, strain JC15 type strain.
SEQ ID NO:51 is Genbank Accession No. AJ874353 Vibrio natriegens partial 16S rRNA gene, strain 01/252.
SEQ ID NO:52 is Genbank Accession No. AF011335 Shewanella pealeana 16S ribosomal RNA gene, complete sequence type strain.
SEQ ID NO:53 is Genbank Accession No. FJ041083 Alteromonadales bacterium fav-2-10-05 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:54 is Genbank Accession No. AY170366 Shewanella waksmanii 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:55 is Genbank Accession No. AJ000214 Shewanella baltica NCTC10735 16S rRNA gene type strain.
SEQ ID NO:56 is Genbank Accession No. AY190533 Shewanella gaetbuli 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:57 is Genbank Accession No. AB094597 Shewanella surugensis gene for 16S rRNA, partial sequence, strain: c959 type strain.
SEQ ID NO:58 is Genbank Accession No. AB094598 Shewanella kaireitica gene for 16S rRNA, partial sequence, strain: c931 type strain.
SEQ ID NO:59 is Genbank Accession No. AF500075 Shewanella pacifica KMM 3597 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:60 is Genbank Accession No. AY326275 Shewanella donghaensis strain LT17 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:61 is Genbank Accession No. AY351983 Shewanella affinis 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:62 is Genbank Accession No. AJ551089 Shewanella psychrophila partial 16S rRNA gene, type strain WP2T.
SEQ ID NO:63 is Genbank Accession No. AJ551090 Shewanella piezotolerans WP3 partial 16S rRNA gene, type strain WP3T.
SEQ ID NO:64 is Genbank Accession No. AY445591 Shewanella profunda strain LT13a 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:65 is Genbank Accession No. AJ609571 Shewanella decolorationis partial 16S rRNA gene, type strain CCTCC M 203093T.
SEQ ID NO:66 is Genbank Accession No. AY485224 Shewanella marisflavi 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:67 is Genbank Accession No. AY485225 Shewanella aquimarina 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:68 is Genbank Accession No. AY579749 Shewanella canadensis strain HAW-EB2 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:69 is Genbank Accession No. AY579750 Shewanella sediminis strain HAW-EB3 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:70 is Genbank Accession No. AY579751 Shewanella halifaxensis strain HAW-EB4 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:71 is Genbank Accession No. AY579752 Shewanella atlantica strain HAW-EB5 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:72 is Genbank Accession No. AF145921 Shewanella sp. KMM3299 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:73 is Genbank Accession No. AY653177 Shewanella colwelliana 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:74 is Genbank Accession No. AB201475 Shewanella abyssi gene for 16S rRNA, partial sequence, strain: c941 type strain.
SEQ ID NO:75 is Genbank Accession No. AB205566 Shewanella hafniensis gene for 16S rRNA, partial sequence, strain: P010 type strain.
SEQ ID NO:76 is Genbank Accession No. AB205570 Shewanella algidipiscicola gene for 16S rRNA, partial sequence, strain: S13 type strain.
SEQ ID NO:77 is Genbank Accession No. AB205571 Shewanella glacialipiscicola gene for 16S rRNA, partial sequence, strain: T147 type strain.
SEQ ID NO:78 is Genbank Accession No. AB205576 Shewanella morhuae gene for 16S rRNA, partial sequence, strain: U1417 type strain.
SEQ ID NO:79 is Genbank Accession No. AB204519 Shewanella pneumatophori gene for 16S rRNA, partial sequence type strain.
SEQ ID NO:80 is Genbank Accession No. DQ167234 Shewanella spongiae strain HJ039 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:81 is Genbank Accession No. DQ180743 Shewanella irciniae strain UST040317-058 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:82 is Genbank Accession No. DQ286387 Shewanella loihica strain PV-4 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:83 is Genbank Accession No. AF295592 Shewanella sp. ACEM-9 16S ribosomal RNA gene, partial sequence type strain.
SEQ ID NO:84 is Genbank Accession No. CP000961 Shewanella woodyi strain MS32=ATCC 51908 REGION: 53358 . . . 54894 type strain.
SEQ ID NO:85 is Genbank Accession No. AP009048 E. coli str. K12 substr. W3110 DNA, complete genome, REGION: 3468481 . . . 3470022.
SEQ ID NO:86 is Genbank Accession No. AE014299 Shewanella oneidensis MR-1 REGION: 46116 . . . 47644.
SEQ ID NO:87 is Genbank Accession No. NC—009438 Shewanella putrefaciens CN-32 REGION: 38741 . . . 40295.
SEQ ID NO:88 is Genbank Accession No. NC—009052 Shewanella baltica OS155 chromosome, complete genome REGION: 44440 . . . 45976.
SEQ ID NO:89 is Genbank Accession No. CP000821 Shewanella sediminis HAW-EB3, complete genome region 51967-53501.
SEQ ID NO:90 is Genbank Accession No. X81622 Shewanella algae 16S rRNA gene (FeRed).
SEQ ID NO:91 is Genbank Accession No. FJ866783 Shewanella algae strain PSB-05 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:92 is Genbank Accession No. HQ851081 Rhodobacter capsulatus strain NBY31 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:93 is Genbank Accession No. FJ866781 Shewanella algae strain PSB-04 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:94 is Genbank Accession No. GU223381 Shewanella sp. EM0501 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:95 is Genbank Accession No. HM016084 Shewanella sp. KJW27 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:96 is Genbank Accession No. EU817498 Alishewanella jeotgali strain MS1 16S ribosomal RNA gene, partial sequence.
SEQ ID NO:97 is Genbank Accession No. X82147 A. rubra 16S rRNA gene (ATCC 29570T).
Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of patent Procedure:







TABLE 1







INFORMATION ON DEPOSITED STRAINS










International



Depositor Identification
Depository



Reference
Designation
Date of Deposit





Shewanella sp L3:3
ATCC No. PTA-10980
May 19, 2010


Shewanella putrefaciens
ATCC No. PTA-8822
Dec. 4, 2007


LH4:18




Shewanella algae MPHPW-1
ATCC No. PTA-11920
Jun. 3, 2011









DETAILED DESCRIPTION
Applicants specifically incorporate the entire content of all cited references in this disclosure. Unless stated otherwise, all percentages, parts, ratios, etc., are by weight. Trademarks are shown in upper case. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
The invention relates to identification of a new strain of Shewanella isolated from production water samples obtained from an oil reservoir and to methods for altering the interfacial properties of hydrocarbon coated surfaces by contacting these surfaces with these Shewanella microorganisms that have the ability to alter the interface between the hydrocarbons and such surfaces. These alterations result in substantial oil liberation from the hydrocarbon-coated surfaces.
The following definitions are provided for the special terms and abbreviations used in this application:
The abbreviation “dNTPs” refers to Deoxyribonucleotide triphosphates.
The abbreviation “ATCC” refers to American Type Culture Collection International Depository, Manassas, Va., USA. “ATCC No.” refers to the accession number to cultures on deposit with ATCC.
The abbreviation “ASTM” refers to the American Society for Testing and Materials.
The term “terrestrial subsurface formation” or “subsurface formation” refers to in ground or under ground geological formations and may comprise elements such as rock, soil, brine, sand, shale, clays and mixtures thereof.
The term “terrestrial surface formation” or “surface formation” refers to above ground geological formations and may comprise elements such as rock, soil, brine, sand, shale, clays and mixtures thereof.
The term “environmental sample” means any sample exposed to hydrocarbons, including a mixture of water and oil. As used herein environmental samples include water and oil samples that comprise indigenous microorganisms useful for phylogenetic mapping of genera present in a given sampling area.
The term “environmental site” means a site that has been contaminated with hydrocarbons and/or other persistent environmental pollutants. Environmental sites may be in surface or subsurface locations.
“Production wells” are wells through which oil is withdrawn from an oil reservoir. An oil reservoir or oil formation is a subsurface body of rock having sufficient porosity and permeability to store and transmit oil. Production fluid containing a mixture of water and oil is also recovered from a production well.
The term “sweep efficiency” refers to the fraction of an oil-bearing stratum that has seen fluid or water passing through it to move oil to production wells. One problem that can be encountered with waterflooding operations is the relatively poor sweep efficiency of the water, i.e., the water can channel through certain portions of the reservoir as it travels from the injection well(s) to the production well(s), thereby bypassing other portions of the reservoir. Poor sweep efficiency may be due, for example, to differences in the mobility of the water versus that of the oil, and permeability variations within the reservoir which encourage flow through some portions of the reservoir and not others.
The term “injection water” refers to fluid injected into oil reservoirs for secondary oil recovery. Injection water may be supplied from any suitable source, and may include, for example, sea water, brine, production water, water recovered from an underground aquifer, including those aquifers in contact with the oil, or surface water from a stream, river, pond or lake. As is known in the art, it may be necessary to remove particulate matter including dust, bits of rock or sand and corrosion by-products such as rust from the water prior to injection into the one or more well bores. Methods to remove such particulate matter include filtration, sedimentation and centrifugation.
The term “irreducible water saturation” is the minimal water saturation that can be achieved in a porous core plug when flooding with oil to saturation. It represents the interstitial water content of the matrix where the water is never completely displaced by the oil because a minimal amount of water must be retained to satisfy capillary forces.
The term “growing on oil” means the microbial species are capable of metabolizing hydrocarbons or other organic components of crude petroleum as a nutrient to support growth.
The term “remediation” refers to the process used to remove hydrocarbon contaminants from an environmental site containing hydrocarbons and/or other persistent environmental pollutants.
The term “bioremediation” refers to the use of microorganisms to remediate or detoxify contaminants form a contaminant-altered environment
“Petroleum” or “oil” is a naturally occurring, flammable liquid found in rock and sand formations in the Earth, which consisting of a complex mixture of hydrocarbons and polycyclic aromatic hydrocarbon of various molecular weights, plus other organic compounds.
“Crude oil” refers to the unrefined oil taken from a petroleum reservoir.
“Oil well” and “oil reservoir” may be used herein interchangeably and refer to a subsurface formation from which oil may be recovered.
“Interface” as used herein refers to the surface of contact or boundary between immiscible materials, such as oil and water or a liquid and a solid. As used herein “interfaces” may be between a water layer and an oil layer, a water layer and a solid surface layer, or an oil layer and a solid surface layer.
“Hydrocarbon-coated” as used herein refers to a coating of a hydrocarbon to a solid surface of at least 10% areal coverage.
The term “components of a subsurface formation” refers to rock, soil, brine, sand, shale, clay or mixtures thereof of either subterranean or seabed formations, that have come in contact with one or more hydrocarbon. These components may be part of an oil well or reservoir. At least a portion of the components include some hydrocarbon-coated surfaces, including particles with coated surfaces.
“Adhered to” refers to coating or adsorption of a liquid to a solid surface of at least 10% areal coverage.
“Shewanella species” or “Shewanella sp.” is a bacterial genus that has been established, in part through phylogenetic classification by rDNA. There is at least about 89% sequence identity of 16S rDNA sequences among Shewanella species. The 16S rDNA sequences of Shewanella species have at least about 89% sequence identity to any of SEQ ID NOs:3-12. Shewanella species have 16S rDNA which has the dominant and degenerate signature sequences listed respectively of regions 2 (SEQ ID NO:18, 19), 5, (SEQ ID NO:20, 21) and 8 (SEQ ID NO: 22, 23) as shown in FIG. 18. The degenerate signature sequence for each region gives the sequence that defines Shewanella species, including some position variations as shown in FIG. 18. The dominant signature sequences in FIG. 18 are those with the variable positions designated as the most frequently found nucleotides in Shewanella species. Additional Shewanella species have the degenerate signature sequence for region 2 of SEQ ID NO:28, replacing SEQ ID NO:19. This sequence has a C in position 23 in the region 2 sequence.
Shewanella are gram negative, gamma-proteobacteria, which have the ability to reduce metals and are capable of additionally reducing a wide range of terminal electron acceptors. These microorganisms gain energy to support anaerobic growth by coupling the oxidation of H2 or organic matter to the redox transformation of a variety of multivalent metals, which leads to the precipitation, transformation, or dissolution of minerals.
The abbreviation “rDNA” refers to ribosomal deoxyribonucleic acid gene sequence.
The term “rDNA typing” means the process of using the sequence of the 16S rDNA gene to obtain the “closest relative” microbial species by homology to rDNA sequences maintained in several international databases.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparisons. In the art, “identity” also means the degree of sequence relatedness or homology between polynucleotide sequences, as determined by the match between strings of such sequences and their degree of invariance. The term “similarity” refers to how related one nucleotide or protein sequence is to another. The extent of similarity between two sequences is based on the percent of sequence identity and/or conservation. “identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in “Computational Molecular Biology, Lesk, A. M., ed. Oxford University Press, NY, 1988”; and “Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NY, 1993”; and “Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NJ, 1994”; and “Sequence Analysis in Molecular Biology, von Heinje, G., ed., Academic Press, 1987”; and “Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, NY, 1991”. Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs such as sequence analysis software. Typical sequence analysis software includes, but is not limited to: the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215, 403-410, 1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, W. R., Comput. Methods Genome Res., Proc. Int. Symp, Meeting Date 1992, 111-120, Eds: Suhai, Sandor, Plenum Publishing, New York, N.Y., 1994). Within the context of this application, it will be understood that, where sequence analysis software is used for analysis, the results of the analysis will be based on the “default values” of the program used, unless otherwise specified. As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.
The term “wetting” refers to the ability of a liquid to maintain contact with a solid surface, resulting from intermolecular interactions when the two are brought together. The degree of wetting (expressed as “wettability”) is determined by a force balance between adhesive and cohesive forces.
“Wetting agent” refers to a chemical such as a surfactant that increases the water wettability of a solid or porous surface by changing the hydrophobic surface into one that is more hydrophilic. Wetting agents help spread the wetting phase (e.g., water) onto the surface thereby making the surface more water wet.
“Wettability” refers to the preference of a solid to contact one liquid, known as the wetting phase, rather than another. Solid surfaces can be water wet, oil wet or intermediate wet. “Water wettability” pertains to the adhesion of water to the surface of a solid. In water-wet conditions, a thin film of water coats the solid surface, a condition that is desirable for efficient oil transport.
The term “adhesive forces” refers to the forces between a liquid and solid that cause a liquid drop to spread across the surface.
The term “cohesive forces” refers to forces within the liquid that cause the drop to ball up and avoid contact with the surface.
The term “contact angle” is the angle at which a liquid (oil or water) interface meets a solid surface, such as sand or clay. Contact angle is a quantitative measurement of the wetting of a solid by a liquid and is specific for any given system, and is determined by interactions across three interfaces. The concept is illustrated with a small liquid droplet resting on a flat horizontal solid surface. The shape of the droplet is determined by the “Young Relation” (Bico et al., Colloids and Surfaces A: Physicochemical and Engineering Aspects 206 (2002)41-46). The theoretical description of contact arises from the consideration of a thermodynamic equilibrium between the three phases: the liquid phase of the droplet (L), the solid phase of the substrate (S), and the gas/vapor phase of the ambient (V) (which will be a mixture of ambient atmosphere and an equilibrium concentration of the liquid vapor). The V phase could also be another (immiscible) liquid phase. At equilibrium, the chemical potential in the three phases should be equal. It is convenient to frame the discussion in terms of interfacial energies. The solid-vapor interfacial energy (see surface energy) is γSV, the solid-liquid interfacial energy is γSL L and the liquid-vapor energy (i.e. the surface tension) is simply γ. The Young equation: 0=γSV−γSL−cos θ is written such that describes an equilibrium where θC is the equilibrium contact angle.
“Microbial populations” means one or more populations of microorganisms present, either in samples obtained from oil wells or in an inoculum for injection into an oil well or subsurface formation.
“Medium” as used herein means an aqueous milieu which supports the growth of organisms. Medium containing Shewanella sp. cells is referred to herein as “cell containing” medium and medium that has been in contact with one or more Shewanella sp. but is a cell free supernatant is referred to herein as “conditioned” medium. Medium will be aqueous based and may contain various nutrients, buffers, salts, vitamins, co-factors and the like and carbon sources useful for microbial growth.
An “electron acceptor” is a molecular compound that receives or accepts an electron during cellular respiration. Microorganisms obtain energy to grow by transferring electrons from an “electron donor” to an electron acceptor. During this process, the electron acceptor is reduced and the electron donor is oxidized. Examples of acceptors include oxygen, nitrate, fumarate, iron (III), manganese (IV), sulfate or carbon dioxide. Sugars, low molecular weight organic acids, carbohydrates, fatty acids, hydrogen and crude oil or its components such as petroleum hydrocarbons or polycyclic aromatic hydrocarbons are examples of compounds that can act as electron donors.
“Denitrifying” and “denitrification” mean reducing nitrate for use as an electron acceptor in respiratory energy generation. “Denitrifying conditions” means conditions where denitrification occurs.
“Inoculating an oil well” means injecting one or more microorganisms or microbial populations or a consortium into an oil well or oil reservoir such that microorganisms are delivered to the well or reservoir without loss of total viability.
The term “simple nitrates” and “simple nitrites” refer to nitrite (NO2) and nitrate (NO3).
The term “nutrient supplementation” refers to the addition of nutrients that benefit the growth of microorganisms that are capable of using crude oil as their main carbon source but grow optimally with other non-hydrocarbon nutrients, i.e., yeast extract, peptone, succinate, lactate, formate, acetate, propionate, glutamate, glycine, lysine, citrate, glucose, pyruvate and vitamin solutions.
The term “biofilm” means a film or “biomass layer” made up of a matrix of a compact mass of microorganisms consisting of structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances. Biofilms are often embedded in these extracellular polymers, which adhere to surfaces submerged in, or subjected to, aquatic environment
The term “bacterial” means belonging to the bacteria. Bacteria are an evolutionary domain or kingdom of microbial species separate from other prokaryotes based on their physiology, morphology and 16S rDNA sequence homologies.
“Microbial species” means distinct microorganisms identified based on their physiology, morphology and phylogenetic characteristics using 16S rDNA sequences. The closest relative microbial species may also be referred to as a “homolog”.
The term “pure culture” means a culture derived from a single cell isolate of a microbial species. The pure cultures specifically referred to herein include those that are publicly available in a depository. Additional pure cultures are identifiable by the methods described herein.
The term “simulated injection brine” or “SIB” is a medium composition containing 198 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 1.2 mM KCl, 16 mM NaHCO3, 0.05 mM SrCl2, 0.13 mM BaCl2, 0.14 mM LiCl.
The abbreviation “NCBI” refers to the National Center for Biotechnology Information.
A spectrophotometer is a device for measuring light intensity that can measure intensity as a function of the color, or more specifically, the wavelength of light. In microbiology, the term “optical density” is a unit-less measure of the transmittance of light at a given wavelength that uses an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling.
The term “MPN” or “most probable number” is a quantitative measurement of the concentration of microbes in a given medium. It is expressed as CFU/ml (colony forming units/ml), log 10 (CFU/ml) or log 10 (MPN).
The term “hypervariable regions” as used herein refers to sequence regions in the 16S rRNA gene where the nucleotide sequence is highly variable. In most microbes the 16S rDNA sequence consists of nine hypervariable regions that demonstrate considerable sequence diversity among different bacterial genera and species and can be used for genus and species identification
The term “signature sequences” as used herein refers to specific nucleotides at specific 16S rRNA encoding gene (rDNA) positions (signature positions), which usually occur within the hypervariable regions, that are distinguishing for microorganisms at different levels. At the signature positions, nucleotides that distinguish between species may be one or more specific base substitutions, insertions or deletions. When taken together, the signature sequences of 16S rDNA are useful for describing microbes at the species, strain or isolate level and can be used in the identification of a microbe.
The term “degenerate base position” refers herein to a position in a nucleotide sequence that may be more than one choice of nucleotide. A position is a “two-fold degenerate” site if only two of four possible nucleotides may be at that position. A position is a “three-fold degenerate” site if three of four possible nucleotides may be at that position. A position is a “four-fold degenerate” site if all four nucleotides may be at that position. “Degeneracy” refers to the existence of more than one nucleotide in at least one position in a specified population of sequences having high identity.
The term “degenerate signature sequence” refers to a signature sequence that has one or more variable position(s) that may include the occurrence of different bases, base insertions and/or base deletions in different individual sequences.
The term “phylogenetic typing” “phylogenetic mapping” or “phylogenetic classification” may be used interchangeably herein and refer to a form of classification in which microorganisms are grouped according to their ancestral lineage. The methods herein are specifically directed to phylogenetic typing on environmental samples based on 16S Ribosomal DNA (rDNA) sequencing. In this context, approximately 1400 base pair (bp) length of the 16S rDNA gene sequence is generated using 16S rDNA universal primers identified herein and compared by sequence homology to a database of microbial rDNA sequences. This comparison is then used to help taxonomically classify pure cultures for use in enhanced oil recovery.
The term “phylogenetics” refers to the field of biology that deals with identifying and understanding evolutionary relationships between organisms, and in particular molecular phylogenetics uses DNA sequence homologies in this analysis. In particular, similarities or differences in 16S rDNA sequences, including signature sequences, identified using similarity algorithms serves to define phylogenetic relationships.
The term “phylogenetic tree” refers to a branched diagram depicting evolutionary relationships among organisms. The phylogenetic tree herein is based on DNA sequence homologies of 16S rDNAs, including of signature sequences in the 16S rDNA, and shows relationships of the present strains to related reference strains and species.
The term “clade” or “phylogenetic clade” refers to a branch in a phylogenetic tree. A clade includes all of the related organisms that are located on the branch, based on the chosen branch point. A clade is defined by a change in one or more signature sequences.
The term “genomovar” is used to describe a sub-species classification which is used when a group of strains of a species are differentiable by DNA sequence, but are phenotypically indistinguishable. Genomovars are defined and identified by DNA-DNA hybridization and/or by 16S rDNA signature sequences. This terminology has been used to describe Pseudomonas stutzeri by Bennasar et al. ((1996) Int. J. of Syst. Bacteriol. 46:200-205).
The term “Ribotyping®” refers to fingerprinting of genomic DNA restriction fragments that contain all or part of the rRNA operon encoding for the 5S, 16S and 23S rRNA genes (Webster, John A, 1988. U.S. Pat. No. 4,717,653; Bruce, J. L., Food Techno., (1996), 50: 77-81; and Sethi, M. R., Am. Lab. (1997), 5: 31-35). Ribotyping® involves generation of restriction fragments, from microbial chromosomal DNA, which are then separated by electrophoresis, and transferred to a filter membrane and finally probed with labeled rDNA operon probes. Restriction fragments that hybridize to the labeled probe produce a distinct labeled pattern or fingerprint/barcode that is unique to a specific microbial strain. Ribotyping® is performed using the DuPont RIBOPRINTER® system.
The term “Riboprint®” refers to the unique genomic fingerprint of a specific microbial isolate or strain, generated using the DuPont RIBOPRINTER® system that can be given a unique “Riboprint® identifier” (alphanumeric characters) and stored electronically to be used to identify the isolate when compared to the database at a later date. The Ribotyping® procedure can be entirely performed on the Riboprinter® instrument (DuPont Qualicon, Wilmington, Del.).
The term “Riboprint® batch” refers to comparison alignment of two or more Riboprints® and is depicted in a report as a pictograph.
The term “type strain” refers to the reference strain recognized in the International Journal of Systematic and Evolutionary Microbiology for a particular species, whose description is used to define and characterize that species. It is one of the first strains of a species studied and is usually more fully characterized than other strains; however, it does not have to be the most representative member. Type strains are usually deposited in a national strain collection, such as the ATCC (USA) or DSMZ (German).
The term “reference strain or species” refers to any strain or species in the public domain.
The term “standard strain” means a strain which matches the type strain in character and sequence. These strains were used as sequence standards in defining signature sequences because their genomes have been completely sequenced and these sequences scrutinized for accuracy
The abbreviation “LPNSN” refers to the List of Prokaryotic names with Standing in Nomenclature (LPNSN), the official database of accepted prokaryote names that have conformity within the rules of the International Code of Nomenclature of Bacteria and have been validated by the International Journal of Systematic and Evolutionary Microbiology.
The acronym “IPOD” refers to International Patent Organism Depository, Independent Administrative Agency National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi 1-chrome, Tsukuba-shi, Ibaraki-ken, Japan), which is a collection of microorganisms.
The acronym “CCUG” refers to the Culture Collection of the University of Göteborg, Sweden, which is a collection of microorganisms.
The terms “water saturation” and “oil saturation” refer to, respectively, the percentage of void volume occupied by water or oil.
“Void volume” is a measure of empty space in a material that is accessible by fluids flowing through that material.
Altering Hydrocarbon-Surface Interface
Provided herein are methods for oil recovery and remediation which rely on altering the wettability of hydrocarbon-coated surfaces. Through altering wettability, the characteristics of the interface between hydrocarbons and a hydrocarbon-coated surface are changed, thereby releasing the hydrocarbons from the surface. For example, this alteration may result in the surface having a preference for binding water as opposed to oil thereby providing for recovery of the oil more readily. Changes in the wettability may be monitored by measuring changes in the contact angle between a hydrocarbon and the surface to which it is adhered. For example, an increase in the contact angle is an indication of a reduction in the surface energy required to bind the oil to the surface (see Example 8). Thus, an aspect of the present invention is the discovery that contact between Shewanella sp. or biomolecules produced by Shewanella sp., and hydrocarbon coated surfaces produces alterations in the wettability properties of the hydrocarbon coated surface such that the surface energy binding the hydrocarbon to the surface is decreased, (as measured by an increase in the contact angle) resulting in the subsequent release of oil.
Hydrocarbon-coated surfaces may be any hard surface (including one or more particles) that is coated or contaminated with hydrocarbons with at least 10% areal coverage by said hydrocarbons. The hydrocarbons may be adhered to said surfaces. Hydrocarbon-coated surfaces may be in subsurface formations, for example in oil reservoirs, and may include rock, soil, brine, sand, clays, shale, and mixtures thereof. In addition, hydrocarbon-coated surfaces may include materials that are not subsurface including rock, clay, soil, sediments, sand, sludge, harbor dredge spoils, sediments, refinery wastes, wastewater, sea water, and mixtures thereof. In addition, hydrocarbon-coated surfaces may include equipment such as pipelines, oil tanks and tankers, and other machinery that may be contaminated with hydrocarbons.
In the present methods, Shewanella sp. alter the wettability of hydrocarbon-coated surfaces. Alteration may be by contact with said microbes or by contact with extracellular molecules produced by said microbes, which may include one or more wetting agents. The Shewanella sp. under certain conditions undergo modifications in surface-bound or extracellular molecules. Modifications include changes in the composition and/or ratio of the molecules which include but are not limited to cytochromes, flavins, siderophores, membrane vesicles, glycoproteins, glycolipids, lipoproteins, fimbriae, extracellular polymeric substances, polysaccharides, monosaccharides, and lipopolysaccharides. These modifications, in turn, alter the water wettability of a hydrocarbon-coated surface contacted by the altered Shewanella microbe.
Conditions for growth that are suitable for Shewanella species to be used in the present methods are determined by the environment of the target hydrocarbon-coated surface, and the conditions for growth of said species in a given environment. Suitable conditions include those that are favorable to producing changes in the wettability of the hydrocarbon-coated surface. Such suitable conditions may include growth and medium compositions that are beneficial for the production and/or modification of surface bound or extracellular molecules, especially those molecules related to stress, oxygen limitation, redox, and/or electron transfer which may be wetting agents. Typical growth media compositions include enriched media containing diverse nutrient sources such as peptone, yeast extract, or casamino acids, for example. In some aspects the media may be a minimal media such as SL10 or simulated injection brine supplemented with an electron donor and electron acceptor. Examples of electron donors include, but are not limited to, lactate and/or acetate. Examples of electron acceptors include but are not limited to, nitrate, fumarate, pyruvate, ferric ion (Fe (III)) and/or manganese ion (Mn (IV). Additional carbon sources may include but are not limited to yeast extract, peptone, pyruvate, glucose, succinate, formate, propionate, glutamate, glycine lysine, oil, and oil components. Oil components may be any of the many components that are present in crude oil. Cultures may be grown aerobically or anaerobically, and may be grown at a temperature that is similar to that of a target reservoir, typically about 30° C., or in the range of room temperature, +/−5° C. In addition, stress conditions may be suitable for growth of the present strains. Growth under stress inducing conditions includes, but is not limited to, switching growth from oxic to anoxic conditions, growth under population pressure or high density growth, switching electron acceptors, growth at low temperatures, and growth under osmotic stress (such as in high salt).
Cultures of Shewanella species may be used to contact hydrocarbon-coated surfaces in the present methods. Alternatively, cells may be removed from the cultures and the remaining medium, which has been conditioned by growth of Shewanella species cells, may be used to contact hydrocarbon-coated surfaces in the present methods. It is likely that conditioned medium contains biosurfactants or other biomolecules that act as wetting agents and contribute to the alterations in the wettability hydrocarbon coated surfaces.
Multiple cultures of different strains of Shewanella species may be used in the present methods. Alternatively, multiple strains may be grown in the same culture that is used in the present methods.
Treating Surface and Subsurface Formations
In the present methods, hydrocarbon-coated surfaces in surface and subsurface formations are contacted with a Shewanella species culture as a cell containing medium or a conditioned medium. Typically the subsurface formations will be contained within an oil well site, often comprising an injection well site and a production well site.
Application of the medium may include processes such as waterflooding, or the use of a fluid such as an aqueous solution or gas (such as CO2) or a solvent or a polymer that is injected into the subsurface formation. Injection methods are common and well known in the art and any suitable method may be used. For example, Nontechnical guide to petroleum geology, exploration, drilling, and production, 2nd edition. N. J. Hyne, PennWell Corp. Tulsa, Okla., USA, Freethey, G. W., Naftz, D. L., Rowland, R. C., &Davis, J. A. (2002); Deep aquifer remediation tools: Theory, design, and performance modeling, In: D. L. Naftz, S. J. Morrison, J. A. Davis, & C. C. Fuller (Eds.); and Handbook of groundwater remediation using permeable reactive barriers (pp. 133-161), Amsterdam: Academic Press.
Typically the injection site or well will communicate with the production well where oil is recovered. The application of the medium (either cell containing or cell free, conditioned) may follow any number of sequences for the effective production of oil and the various options will be readily apparent to the one skilled in the art of oil recovery.
For example treatment of the subsurface formations may include pumping or adding water containing Shewanella microbes via an injection well into an area comprising hydrocarbons (“treatment zone”) and allowing that water to be produced along with the recovered hydrocarbon at the production well. Treatment may also involve pumping water with cell-free medium produced by conditioning with Shewanella into a treatment zone. Treatment of an oil reservoir also may include pumping water with medium down the producer well and into the formation and then back-flowing oil and water out of the producer well (huff and puff). Additionally reservoir treatment may also include inoculating an injector well that is in communication with one or more producer wells, and then subsequently adding an injection water that has been augmented with nutrients either continuously or periodically to promote growth of the Shewanella microbes, where oil is recovered at the producer well. Other treatments may include pumping water containing conditioned medium onto an environmental site comprising elements as a pile of oil sand or oil shale, collecting the water and released oil, and separating the oil from the water. The water may optionally be recycled back to be treated with Shewanella sp.
Hydrocarbon-coated surfaces may be contacted with cell containing Shewanella sp. medium or conditioned medium alone or with additional components. Additional components may be provided separately or in compositions with the medium. Components other than cultures may be injected, pumped, or otherwise applied to an area with hydrocarbon-coated surfaces prior to, together with, or following contact with cultures or conditioned medium.
Mixtures of the present one or more Shewanella species and at least one electron acceptors provide compositions for use in any oil recovery or clean-up site as listed above. Electron acceptors may include, but are not limited to, nitrate, fumarate, pyruvate, ferric ion (Fe (III)) or manganese ion (Mn (IV)). Mixtures of one or more electron acceptor may be used.
Additional components of the compositions may include at least one carbon sources, such as but not limited to, lactate, yeast extract, peptone, pyruvate, glucose, succinate, formate, acetate, propionate, glutamate, glycine lysine, oil, and oil components. Oil components may be any of the many components that are present in crude oil.
The compositions may include other agents or components such as one or more additional microorganisms, such as bacteria, yeast, or fungus. Particularly useful additional microorganisms are capable of growing on oil under denitrifying conditions. In some embodiments, the additional agents may be the microorganisms Pseudomonas stutzeri strain LH4:15 (ATCC No. PTA-8823), and/or Thauera sp AL9:8, (ATCC No. PTA-9497), which are described in commonly owned and co-pending US 20090263887 A1, and U.S. Pat. No. 7,708,065. Other agents may also include one or more chemical compounds that are not lethal to microorganisms, but are effective at degrading or partially degrading hydrocarbons and/or other contaminants.
Enhanced Oil Recovery from a Reservoir or Oil Well
Enhanced oil recovery in this context may include secondary or tertiary oil recovery of hydrocarbons from subsurface formations by techniques that alter the original properties of hydrocarbon-coated surface interface. Specifically, hydrocarbons that are adhered to surfaces within subsurface formations may be substantially liberated by contact with Shewanella sp. or biomolecules produced by these microorganisms. Typically oil is liberated on an order of about 5, 10, 15, 20, 25, 30, to about 35% of the areal coverage. These methods permit the release of oil that could not normally be recovered by waterflooding or other traditional oil recovery techniques.
Bioremediation.
In addition to applications in oil recovery the present Shewanella sp may be useful in effecting the remediation of environmental sites contaminated with hydrocarbons and other pollutants. Bioremediation strategies for hydrocarbons depend on their locations in the environment. Contamination by hydrocarbon spills can be costly to remediate and cause toxicity to environmental inhabitants. Use of microbial action as described here may provide cost-effective mechanisms for remediating hydrocarbon contamination especially under circumstances in which contamination results in hydrocarbon-coated surfaces. For example, use of Shewanella sp. and their surface active agents (such as wetting agents) help to increase wettability of soil and solubility of soil contaminants through reduction in surface and interfacial tensions. This action liberates the hydrocarbons from the surface of soils and renders them available for other remediating action, including degradation by other microbes. In this context bioremediation may be accomplished by a combination of microbes including Shewanella sp. in addition to oil-degrading microorganisms.
Shewanella Species
It has been discovered that the presence of Shewanella species, or materials or biomolecules produced by Shewanella, has the effect of altering the wettability of a hydrocarbon coated surface. Any and all members of the genus Shewanella have this utility.
Shewanella is a bacterial genus that has been established, in part through phylogenetic classification by rDNA and is fully described in the literature (see for example Fredrickson et al., Towards Environmental Systems Biology Of Shewanella, Nature Reviews Microbiology (2008), 6(8), 592-603; Hau et al., Ecology And Biotechnology Of The Genus Shewanella, Annual Review of Microbiology (2007), 61, 237-258).
It is within the scope of the present invention to classify relevant Shewanella on the basis of conserved regions contained in the 16S rDNA. Analysis of the 16 S rDNA from 50 different Shewanella strains revealed three conserved signature regions each having dominant and degenerate sequences listed respectively: 2 (SEQ ID NO:18, 19), 5, (SEQ ID NO:20, 21) and 8 (SEQ ID NO:22, 23) as shown in FIG. 18.
To identify the Shewanella signature sequences, 50 different 16S rDNA sequences of Shewanella strains that are available in the NCBI database were aligned. The sequences are from strains that have been classified as Shewanella in the International Journal of Systematic and Evolutionary Microbiology. The sequences were aligned using the MegAlign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the “Clustal method of alignment” (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. In addition to the Shewanella rDNA sequences, the alignment included 16S rDNA sequences of E. coli (SEQ ID NO:85), and of microbes closely related to Shewanella: Alishewanella jeotgali (SEQ ID NO:96), Pseudoalteromonas rubra (SEQ ID NO:97), and Vibrio natriegenas (SEQ ID NO:51). Through visual analysis of the 16S rDNA variable regions 2, 5, and 8, signature sequences for Shewanella species were identified and are given in FIG. 18.
In further analysis of the 16S rDNA sequences of 55 recognized species of Shewanella from the List of Prokaryotic names with Standing in Nomenclature (LPNSN), as described in Example 26 herein, an additional base variation was identified for Shewanella strains that is in position 23 of region 2. The sequence for region 2 with the new base variation of C at position 23 and degeneracy at other positions that are in SEQ ID NO:19 is SEQ ID NO:28, and the degenerate sequence for region 2 that includes this variation in position 23 by placing V at this position (V=A/C/G) is SEQ ID NO:25. Thus Shewanella sp. useful in the present invention are those that comprise within the 16s rDNA the dominant or degenerate signature sequences for each of regions 2, 5, and 8 as set forth in SEQ ID NOs:18-23, as well as those with SEQ ID NO:25 and 28 for region 2 instead of SEQ ID NO:18 or 19. Specifically, Shewanella sp. comprising 16S rDNA comprising SEQ ID NOs:21, 23, and 28 may be used in the present invention.
Specific strains of Shewanella are disclosed herein that are useful in the methods of the invention. One such strain is Shewanella putrefaciens strain LH4:18 which was isolated, identified, and deposited to the ATCC under the Budapest Treaty as #PTA-8822, as described in commonly owned and co-pending U.S. Pat. No. 7,776,795. Strain LH4:18 has the 16S rDNA sequence of SEQ ID NO:5.
Examples of additional Shewanella species that may be used include but are not limited to Shewanella frigidimarina (DSM 12253), Shewanella pacifica (DSM 15445), Shewanella profunda (DSM 15900), Shewanella gelidimarina (DSM 12621), and Shewanella baltica (DSM 9439). These strains may be purchased through the German Collection of Microorganisms and Cell Cultures (DSMZ). These and other strains that may be used have at least about 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5 of strain LH4:18.
Additionally useful strains are Shewanella strains EH60:12, EH60:2, and EH60:10, which were identified herein and characterized with partial 16S rDNA sequences of SEQ ID NOs:15, 16, and 17, respectively. Shewanella species include microorganisms having a 16S rDNA sequence with at least about 95%, 96%, 97%, 98%, or 99% identity to any one or all of SEQ ID NOs:15-17.
In addition to the known Shewanella sp. described above, a newly identified Shewanella sp. which is useful in the present methods was disclosed in US Pat. Appl. Pub. #2011-0030956 A1. This new strain is identified as Shewanella sp. strain L3:3 which has been deposited with the ATCC under the Budapest Treaty as #PTA-10980. Shewanella sp. strain L3:3 was isolated from an injection water sample obtained from the Alaskan North Slope and has the 16S rDNA sequence of SEQ ID NO:3. Within the 16S rDNA sequence are signature sequences that were identified in variable regions 3 and 6 of prokaryote rDNA that have nucleotide sequences of SEQ ID NOs: 13 and 14, respectively. As shown in FIG. 14, the nucleotides at specific positions (with respect to the first nucleotide of SEQ ID NO:3) 438-40, 451, 454-57, 466-69, 471, 484-86 and 496 within SEQ ID NO:13 are different in strain L3:3 from the nucleotides present in the 16S rDNA of Shewanella putrefaciens, Shewanella sp. LH4:18 and Shewanella algae. As shown in FIG. 15, the nucleotides at specific positions (with respect to the first nucleotide of SEQ ID NO:3) 995-6, 1001-05, 1007, 1012, 1014, 1016-1018 and 1035 within SEQ ID NO:14 are also different in strain L3:3 from the nucleotides present in the 16S rDNA of Shewanella putrefaciens, Shewanella sp. LH4:18 and Shewanella algae. Shewanella strains found herein to have the same nucleotides at all of these positions are Shewanella sp.C31 (SEQ ID:8), Shewanella sp. L-10 (SEQ ID:9), Shewanella chilikensis JC5T (SEQ ID:10), Shewanella sp. C16-M (SEQ ID:12), and a Shewanella clone identified as D00402024H07 (SEQ ID:11). While having the signature sequences of SEQ ID NOs:13 and 14, the present Shewanella species that are closely related to the newly identified strain L3:3 have at least about 97%, 98% or 99% sequence identity to the DNA sequences for 16S ribosomal RNA of SEQ ID NO:3. In addition, strains closely related to strain L3:3 have a Riboprint® pattern identifier of 212-824-S-4 as demonstrated in FIG. 16. This Riboprint® pattern was identified herein for Shewanella sp. strain L3:3.
Within the 16S rDNA sequence are signature sequences that were identified in variable regions 3 and 6 of prokaryote rDNA that have nucleotide sequences of SEQ ID NOs: 13 and 14, respectively. As shown in FIG. 14, the nucleotides at specific positions (with respect to the first nucleotide of SEQ ID NO:3) 438-40, 451, 454-57, 466-69, 471, 484-86 and 496 within SEQ ID NO:13 are different in strain L3:3 from the nucleotides present in the 16S rDNA of Shewanella putrefaciens, Shewanella sp. LH4:18 and Shewanella algae. As shown in FIG. 15, the nucleotides at specific positions (with respect to the first nucleotide of SEQ ID NO:3) 995-6, 1001-05, 1007, 1012, 1014, 1016-1018 and 1035 within SEQ ID NO:14 are also different in strain L3:3 from the nucleotides present in the 16S rDNA of Shewanella putrefaciens, Shewanella sp. LH4:18 and Shewanella algae. Shewanella strains found herein to have the same nucleotides at all of these positions are Shewanella sp. C31, Shewanella sp. L-10, Shewanella chilikensis JC5T, Shewanella sp. C16-M, and a Shewanella clone identified as D00402024H07. While having the signature sequences of SEQ ID NOs:13 and 14, the present Shewanella species that are closely related to the newly identified strain L3:3 have at least about 97%, 98% or 99% sequence identity to the DNA sequences for 16S ribosomal RNA of SEQ ID NO:3.
The present invention provides a newly identified strain of Shewanella which is useful in the present methods. This new strain is identified as Shewanella algae strain MPHPW-1 which was isolated, identified, and deposited to the ATCC under the Budapest Treaty as #PTA-11920. The new strain named MPHPW-1 was isolated from oil well production water collected from an oil reservoir near Wainwright, Alberta Canada. The 16S rDNA sequence of MPHPW-1 was determined to be SEQ ID NO:24.
Strain MPHPW-1 was identified as a Shewanella strain by using 16S rDNA sequence analysis consistent with the criteria set forth in the International Journal of Systematic and Evolutionary Microbiology (B. J. Tindall, R. Rosselló-Mora, H.-J. Busse, W. Ludwig and P. Kämpfer, Int. J. Syst. Evol. Microbiol. 60 (2010), pp. 249-266). A multiple sequence alignment was performed of the MPHPW-1 16S rDNA sequence and the 16S rDNA sequences of the type strains of 55 recognized species of Shewanella from the List of Prokaryotic names with Standing in Nomenclature (LPNSN), as well as a few additional representative strains that are listed in Table 13. This multiple sequence alignment showed that among all pairs of the aligned Shewanella type species sequences there was at least 90% identity with the MPHPW-1 sequence confirming that MPHPW-1 is a Shewanella. 
Of the strains in this alignment, MPHPW-1 is most closely related to the BrY strain that is classified as a Shewanella algae (Venkateswaran, K, et al. International Journal of Systematic Bacteriology (1999) pp. 705-724). This relationship is shown in the phylogenetic tree diagram in FIG. 24, which is described in Example 26 herein. Based on this relationship, the MPHPW-1 strain was identified as Shewanella algae MPHPW-1.
To further characterize strain MPHPW-1, the MPHPW-1 16S rDNA sequence was aligned with all sequences retrieved in a BLAST search having at least as high percent identity as the BrY sequence. Each of these sequences had at least four position differences with the sequence of MPHPW-1, including nucleotide changes, insertions, and deletions. Thus, based on the 16S rDNA sequence analysis, MPHPW-1 was identified as a new strain of Shewanella algae. RiboPrint® analysis confirmed that the genomic sequences surrounding the rDNA operons in strain MPHPW-1 have different genomic structure than those of any strain represented in the RiboPrint® database (7525 patterns contained within DuPont Environmental Services and Qualicon libraries compiled from samples taken from DuPont as well as another 6950 patterns that DuPont Qualicon has supplied from standard identified organisms). Shewanella algae strain BrY, whose sequence identity to strain MPHPW-1 is 99.8%, has a RiboPrint® pattern that is similar to that of MPHPW-1, but its pattern is missing the 1 kb band (fragment). Therefore strain MPHPW-1's RiboPrint® is a unique genomic identifier of the new Shewanella strain MPHPW-1.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art may ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, may make various changes and modifications of the invention to adapt it to various usages and conditions.
Additional Abbreviations Used in the Examples
The meaning of abbreviations is as follows: “hr” means hour(s); “mL” or: ml” means milliliter; “° C.” means degrees Celsius; “mg” means milligram(s); “mm” means millimeter; “g” means gram(s); “GC” means gas chromatography; “g of oil/g of total fluid” means gram of oil per gram of total fluid; “ppm” means part per million; “mM” means millimolar; “%” means percent; “CFU/mL” means colony forming unit per milliliter; :“LB” means Luria broth medium; “min” means minute(s); “mL/min means milliliter per minute; “NIC” means non-inoculated control (negative controls in microbial culture experiments); “μg/L” means microgram per liter; “nM” means nanomolar; “μM” means micromolar, “PSIG” means pounds-force per square inch gauge.
General Methods
Growth of Microorganisms
Techniques for growth and maintenance of anaerobic cultures are described in “Isolation of Biotechnological Organisms from Nature”, (Labeda, D. P. ed. 117-140, McGraw-Hill Publishers, 1990). When using nitrate as an electron acceptor in anaerobic cultures, growth is measured by nitrate depletion from the growth medium over time. Nitrate is utilized as one of the primary electron acceptors under the growth conditions used herein. The reduction of nitrate to nitrogen has been previously described (Moreno-Vivian, C., et al., J. Bacteriol., 181, 6573-6584, 1999). In some cases nitrate reduction processes lead to nitrite accumulation, which is subsequently further reduced to nitrogen. Accumulation of nitrite is therefore also considered evidence for active growth and metabolism by microorganisms.
Determination of Viable Cell Titer (Most Probable Number: MPN)
In order to determine viable cell titer, samples from cultures or sand packs were diluted by 1:10 serial dilution in 8 rows per sample of a 96 well plate using standard Miller's Luria Broth or Luria Broth with 3.5% NaCl added. Titration was done using an automated Biomek200 robotic pipettor. Growth was determined by visual turbidity and recorded for each of 8 rows. The most probable number algorithm of Cochran (Biometrics (1950) 6:105-116) was used to determine the viable cells/ml and the 95% confidence limits for this number in the original sample.
The serial dilution method plating was used to determine the bacterial titer of such cultures. A series of 1:10 dilutions of such samples was plated and the resulting colonies were counted. The number of colonies on a plate was then multiplied by the dilution factor (the number of times that the 1:10 dilution was done) for that plate to obtain the bacterial count in the original sample as CFU/mL.
Ion Chromatography
An ICS2000 chromatography unit (Dionex, Banockburn, Ill.) was used to quantitate nitrate and nitrite ions in growth medium. Ion exchange was accomplished on an AS15 anion exchange column using a gradient of 2 to 50 mM potassium hydroxide. Standard curves were generated and used for calibrating nitrate and nitrite concentrations.
Genomic DNA Extractions from Bacterial Cultures
To extract genomic DNA from liquid bacterial cultures, cells were harvested by centrifugation (10,000 rpm, at room temperature) and resuspended in lysis buffer (100 mM Tris-HCL, 50 mM NaCl, 50 mM EDTA, pH8.0) followed by agitation using a Vortex mixer. Reagents were then added to a final concentration of 2.0 mg/mL lysozyme, 10 mg/mL SDS, and 10 mg/mL Sarkosyl to lyse the cells. After further mixing with a Vortex mixer, 0.1 mg/mL RNAse and 0.1 mg/mL Proteinase K were added to remove RNA and protein contaminants, and the samples were incubated at 37° C. for 1.0-2.0 hr. Post incubation, the samples were extracted twice with an equal volume of a phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) and once with chloroform:isoamyl alcohol (24:1). One-tenth volume of 5.0 M NaCl and two volumes of 100% ethanol were added to the aqueous layer, and mixed. The tubes were frozen at −20° C. overnight and then centrifuged at 15,000×g for 30 min at room temperature to pellet chromosomal DNA. The pellets were washed once with 70% ethanol, centrifuged at 15,000×g for 10 min, dried, resuspended in 100 μL of de-ionized water and stored at −20° C. An aliquot of the extracted DNA was visualized on an agarose gel to ascertain the quantity and quality of the extracted DNA.
Direct Colony rDNA Sequence Analysis
Genomic DNA from bacterial colonies was isolated by diluting bacterial colonies in 50 μL of water or Tris-HCL buffer pH7-8. Diluted colony DNAs were amplified with Phi 29 DNA polymerase prior to sequencing (GenomiPHI Amplification Kit GE Life Sciences, New Brunswick, N.J.). An aliquot (1.0 μL) of a diluted colony was added to 9.0 μL of the Lysis Reagent (from the GenomiPHI Amplification Kit) and heated to 95° C. for 3 min followed by immediate cooling to 4° C. 9.0 μL of Enzyme Buffer and 1.0 μL of Phi 29 enzyme were added to each lysed sample followed by incubation at 30° C. for 18 hr. The polymerase was inactivated by heating to 65° C. for 10 min followed by cooling to 4° C.
DNA sequencing reactions were set up as follows: 8.0 μL of GenomiPHI amplified sample were added to 8.0 μL of BigDye v3.1 Sequencing reagent (Applied Biosystems, Foster City, Calif.) followed by 3.0 μL of 10 μM primers SEQ ID NO: 1 in combination with SEQ ID NO: 2 (prepared by Sigma Genosys, Woodlands, Tex.), 4.0 μL of 5× BigDye Dilution buffer (Applied Biosystems) and 17 μL Molecular Biology Grade water (Mediatech, Inc., Herndon, Va.). Sequencing reactions were heated for 3 min at 96° C. followed by 200 thermocycles of (95° C. for 30 sec; 55° C. for 20 sec; 60° C. for 2 min) and stored at 4° C. Unincorporated fluorescently labeled ddNTPs were removed using Edge Biosystems (Gaithersburg, Md.) clean-up plates. Amplified reactions were pipetted into wells of a pre-spun 96 well clean up plate. The plate was centrifuged for 5 min at 5,000×g in a Sorvall RT-7 (Sorvall, Newtown, Conn.) at 25° C. The cleaned up reactions were placed directly onto an Applied Biosystems 3730 DNA sequencer and sequenced with automatic base-calling. Each of the assembled rDNA sequences was compared to the NCBI rDNA database ( ̃260,000 rDNA sequences) using the BLAST algorithm (Altschul et al., supra). The primary hit was used as an identifier of the most closely related known species identification. The initial screen using the rDNA colony direct sequencing reduced the number of colonies to be carried through further screening by 20 fold.
Automated Ribotyping
Automated ribotyping was used for identification of selected strains with similar 16S rDNA sequence phylogenetic characteristics (Webster, John A, 1988. U.S. Pat. No. 4,717,653; Bruce, J. L., Food Techno., (1996), 50: 77-81; and Sethi, M. R., Am. Lab. (1997), 5: 31-35). Ribotyping was performed as recommended by the manufacturer (DuPont Qualicon Inc., Wilmington, Del.). For these analyses, one fresh colony was picked, resuspended in the sample buffer and added to the processing module for the heat treatment step at 80° C. for 10 min to inhibit endogenous DNA-degrading enzymes. The temperature was then reduced and lytic enzymes lysostaphin and N-acetyl-muramidase, provided by the manufacturer, were added to the sample. The sample carrier was then loaded onto the Riboprinter® system with other commercial reagents. Restriction enzyme digestion using EcoRI enzyme, gel electrophoresis and blotting steps were completely automated. Briefly, bacterial DNA was digested with the EcoRI restriction enzyme and loaded onto an agarose gel, restriction fragments were separated by electrophoresis and then transferred to a nylon membrane. After a denaturation step, the nucleic acids were hybridized with a sulfonated DNA probe containing the genes for the small and large rRNA subunits of E. coli, the 5S, 16S, and 23S ribosomal rRNAs. The hybridized labeled-probe was detected by capturing light emission from a chemiluminescent substrate with a charge-coupled device camera. The output consisted of a densitometry finger scan depicting the specific distribution of the EcoRI restriction fragments containing the genomic rDNA sequences and their molecular weights, which are particular to the genomic DNA sequence of a specific strain independent of the 16S rDNA sequence.
Measuring the Potential for Microbes to Release Oil from Sand Particles
In order to screen test cultures for the ability to release oil from a nonporous silica medium, a microtiter plate assay to measure the ability of the microbes to release oil/sand from oil-saturated North Slope sand was developed. The assay is referred to as the LOOS test (Less Oil On Sand). Autoclaved North Slope sand was dried under vacuum at 160° C. for 48 hr. Twenty grams of the dried sand was then mixed with 5 mL of autoclaved, degassed crude oil. The oil-coated sand was then allowed to age anaerobically at room temperature, in an anaerobic chamber, for at least a week. Microtiter plate assays were set up and analyzed in an anaerobic chamber. Specifically, 2 mL of test cultures were added into the wells of a 12-well microtiter plate (Falcon Multiwell 12 well plates, #353225, Becton Dickinson, Franklin Lakes, N.J.). The control wells contained 2 mL of the medium alone. Approximately 40 mg of oil-coated sand was then added to the center of each well. Samples were then monitored over time for release and accumulation of “free” sand that collected in the bottom of the wells. Approximate diameters (in millimeters) of the accumulated total sand released were measured. A score of 2 mm and above indicates the microbes' potential to release oil from the nonporous silica medium.
Gas Chromatography for Determining Residual Oil on Sand
A gas chromatography (GC) method was developed to analyze the sand from sandpacks for residual oil. An empirical relationship was determined based on the North Slope sand and the intrinsic pore volume of packed sand, e.g., for 240 g of packed sand there is a pore volume of about 64 mL. Weights of the individual sand samples were obtained and the oil on the sand was extracted with a known amount of toluene. A sample of this toluene with extracted oil was then analyzed by GC. A calibration curve was generated and used to determine the amount of oil in toluene on a weight percent basis. This was then multiplied by the total amount of toluene used to extract the oil resulting in the total amount of oil on the sand. This value was then divided by the total sample weight to yield the percent of oil with respect to the total sample weight. The weight percent of oil of the sample was multiplied by the ratio of the empirically derived characteristic of packed North Slope sand (total weight of sample after being flooded with brine divided by total sand weight, 1.27). This relationship was equal to the amount of oil on dry sand. This value was then multiplied by the ratio of the weight of North Slope sand to the weight of fluid trapped in the pore space of the sand, 3.75. This resulting value was the residual oil left on the sand in units of g of oil/g of total fluid in the pore space.
Growth Medium and Growth Protocol
PPGAS medium was used in the following Examples unless stated otherwise. The medium contained: 1.6 mM MgSO4, 20 mM KCl, 20 mM NH4Cl, 120 mM Tris base 0.5% glucose and 1% Bacto peptone. The initial culture was grown aerobically in the medium at 25° C.
Sterile injection brine (SIB) contained: 198 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 1.2 mM KCl, 16 mM NaHCO3, 0.05 mM SrCl2, 0.13 mM BaCl2, 0.14 mM LiCl) plus 1% peptone.
The SL10 medium had the following composition summarized in Table 2 below:







TABLE 2







Composition of the SL10 Medium










Final
Chemical


Growth component
Concentration
Source













Nitrogen
18.7
mM
NH4Cl


Phosphorus
3.7
mM
KH2PO4


Magnesium
984
μM
MgCl2•6H2O


Calcium
680
μM
CaCL2•2H2O


Sodium chloride
172
mM
NaCl


Trace metals






7.5
μM
FeCl2•4H2O



12
nM
CuCl2•2H2O



500
nM
MnCL2• 4H2O



800
nM
CoCl2•6H2O



500
nM
ZnCl2



97
nM
H3BO3



149
nM
Na2MoO4•2H2O



100
nM
NiCl2•6H2O


Selenium-tungstate
22.8
nM
Na2SeO3•5H2O



24.3
nM
Na2WO4•2H2O


pH buffer/Bicarbonate
29.7
mM
NaHCO3


Vitamins
100
μg/L
vitamin B12



80
μg/L
p-aminobenzoic acid



20
μg/L
D(+)-Biotin



200
μg/L
nicotinic acid



100
μg/L
calcium pantothenate



300
μg/L
pyridoxine





hydrochloride



200
μg/L
thiamine-HCL•2H2O



50
μg/L
Alpha-lipoic acid





The pH of the medium was adjusted to between 7.4-7.8.






Example 1
Comparison of the Ability of Early and Late Stage Microbial Cultures to Release Oil from Sand Particles
To determine whether late stationary phase growth enhances oil release, the oil release activity of an anaerobic overnight culture of strain LH4:18 was compared to that of a one week old culture of the same strain. A culture was grown initially as described above in PPGAS medium. It was then moved into an anaerobic chamber, was supplemented with 500 ppm sodium lactate and 1000 ppm sodium nitrate, and divided in half. One half was used immediately in an anaerobic LOOS test, described in General Methods. The other half (Week 1 culture) was aged (left for a week in the anaerobic chamber) and then the LOOS test was performed using this culture.
FIG. 1 shows the relative sand release by strain LH4:18 cultures over a period of three weeks. After about 6 days, a 6 mm zone of released sand was observed in the bottom of the wells for the week old (week 1) culture and a 3 mm zone was observed for the day-old sample (Day 1). Thus these results indicate that late stationary phase growth cultures may be more effective in expression of wetting agent molecules in that the rate of the sand/oil release was higher for the week old sample and continued to increase with time.
Example 2
Demonstration of Oil Release During Both Aerobic and Anaerobic Growth
To ascertain whether oil release occurs when the assay is performed aerobically versus anaerobically, and whether the addition of lactate and nitrate are beneficial, the following experiment was performed. A LOOS test was set up as described above. A culture of strain LH4:18 was grown aerobically overnight at 25° C. in the PPGAS medium. It was then divided in half. One half was supplemented with 1000 ppm sodium lactate and 2000 ppm sodium nitrate. The other half received no further supplements. Each of these cultures was then divided into an aerobic set and an anaerobic set. LOOS tests were set up to compare the samples. PPGAS medium alone samples, with and without the respective supplements, were used as controls.
The results showed that the sand/oil release was relatively the same irrespective of whether the assay was performed aerobically or anaerobically (FIG. 2). Interestingly, the addition of lactate and nitrate had a detrimental effect on both aerobic and anaerobic cultures. It should be noted, however, that even with the aerobic cultures, oxygen could still be limiting due to the high cell density.
Example 3
Comparison of Electron Acceptors and their Effect on Oil Release by Strain LH4:18
A survey of the literature shows that fumarate may act as an efficient terminal electron acceptor (Morris, C. J., et al., Biochem. J., 302: 587-593, 1994). In addition, in Shewanella species, certain cell surface and respiratory molecules are more abundant in cells grown with fumarate, rather than nitrate or iron citrate, as the terminal electron acceptor (Morris et al., supra). In Example 2, it was demonstrated that nitrate addition was detrimental in oil/sand release. Fumarate was therefore tested as an acceptable and possibly more advantageous replacement in this assay.
A frozen stock culture of strain LH4:18 was diluted 1:100 in SIB plus 1% peptone and placed into an anaerobic chamber. The culture was then split and sodium nitrate (2000 ppm), both sodium lactate (1000 ppm) and sodium nitrate (2000 ppm), sodium fumarate (2000 ppm), or both sodium lactate and sodium fumarate supplements were added to different samples. The control sample contained no additional supplements. Samples were grown anaerobically for 3 days. On the second day, samples were fed again with their respective supplements. MPNs were monitored at Day 1 and again after 3 days of anaerobic growth. On Day 3, a LOOS test was set up and sand/oil release was compared across all samples over time.
FIG. 3 shows a comparison of CFU/mL, expressed as Log 10 (MPN), on the day of the LOOS test set up (Day 3) and the relative sand release for each sample. The results show that even though growth was relatively the same across all conditions, the sand/oil release was better for samples containing fumarate instead of nitrate as the terminal electron acceptor.
Example 4
Demonstration of the Effect of Various Media Formulations on Oil Release by Strain LH4:18
For certain bacterial species, glucose is necessary for the expression of some surface molecules and surfactants. To determine whether glucose can improve oil release using strain LH4:18, a LOOS test with this strain grown aerobically overnight in PPGAS medium with and without glucose was performed. The samples were then placed into an anaerobic chamber and the LOOS test was performed anaerobically as described above.
FIG. 4 shows that the sand/oil release response was relatively the same whether glucose was present or not
In order to determine if the effect of strain LH4:18 on oil release was limited to a rich medium, the LOOS test response was measured using different media. Media tested were PPGAS, LB, and supplemented simulated injection brine (SIB). SIB was supplemented with 1% peptone and either MgSO4 and KCl, NH4CL, or Tris. Cultures of strain LH4:18 were grown aerobically overnight. Samples were then placed into an anaerobic chamber and the LOOS test was performed anaerobically.
The simulated injection brine with 1% peptone added worked as well as the other rich medium formulations as shown in FIG. 5. Strain LH4:18 grew relatively the same in each of the media. All cultures exhibited about the same sand/oil release response in the LOOS test.
To determine whether yeast extract worked as well as peptone in the simulated injection brine, these supplements were compared directly in a LOOS assay. Strain LH4:18 was grown aerobically overnight at 25° C. in SIB supplemented with 1% peptone or 1% yeast extract (YE). After 20 hr, the SIB/peptone culture had approximately 4.27E+09 CFU/mL and the SIB/YE culture contained about 9.33E+09 CFU/mL. The samples were then placed into the anaerobic chamber and the LOOS test was performed anaerobically.
The data in FIG. 6 shows that YE may be substituted for peptone with no detrimental effect on the oil release response.
Example 5
Demonstration of Oil Release by Culture Supernatant
A number of microbial species release surfactants in their surrounding media. To determine whether a wetting agent from strain LH4:18 might be released into the surrounding medium, a LOOS test was performed using both a whole LH4:18 culture and also the supernatant alone of an LH4:18 culture. Strain LH4:18 was grown aerobically overnight at 25° C. in SIB supplemented with 1% peptone. After 20 hr, the culture contained approximately 1.49E+09 CFU/mL. The culture was then divided into two aliquots and one aliquot was centrifuged at 12000×g for 3 min to remove the cells. The supernatant was collected from the centrifuged sample and transferred into a new tube. Both samples were then placed into an anaerobic chamber and the LOOS test was performed anaerobically as described above.
FIG. 7 shows that the supernatant alone released the sand/oil as effectively as the whole culture indicating that an agent affecting oil release was present in the medium.
While the Example above showed that the supernatant alone released the sand/oil almost as effectively as the whole culture, an experiment was performed to determine if oil release ability remained surface bound. A culture of strain LH4:18 was grown overnight at 25° C. in SIB supplemented with 1% peptone. The culture was then divided into two aliquots and half was centrifuged at 12000×g for 3 min to remove the cells. The supernatant was collected from the centrifuged sample and transferred into a new tube. The pellet was then resuspended in fresh medium. The other half of the overnight culture was also centrifuged and the supernatant was filtered (Supor, 0.2 μm, Pall Corp., Ann Arbor, Mich.) to remove the microorganisms. The three samples (centrifuged supernatant, filtered supernatant, and resuspended cells) were then placed into the anaerobic chamber and the LOOS test was performed anaerobically. FIG. 8 shows that both supernatant samples released the oil/sand equally well, while oil release by the resuspended cells was less effective. However the resuspended cells were able to cause some oil release.
Example 6
Effect of Strain LH4:18 in Combination with Pseudomonas Stutzeri Strain LH4:15 in Oil Release
To determine whether the oil release effected by strain LH4:18 is compromised by the presence of other microbes, a LOOS test was performed on strain LH4:18 alone and also in the presence of Pseudomonas stutzeri LH4:15 (ATCC No. PTA-8823). Specifically, cultures of strains LH4:15 and LH4:18 were grown separately overnight in the PPGAS medium. Three LOOS tests were performed: 1) using strain LH4:15 alone; 2) using strain LH4:18 alone; and 3) using the combined cultures. The results shown in FIG. 9 indicate that the oil release ability of strain LH4:18 was not adversely affected by the presence of the other microorganism.
Example 7
Measuring the Effects of Other Shewanella Species in Oil Release
Additional Shewanella strains had been identified through anaerobic enrichments on oil production fluids, using SL10 medium and Fe(III) as the electron acceptor. Strains EH60:12, EH60:10, and EH60:2 were identified as Shewanella species by their 16S rDNA sequences (SEQ ID NOs:15, 16, and 17, respectively). These strains were grown aerobically overnight in the LB medium. A LOOS test was set up on 2 mL of the whole cultures as previously described.
The results in FIG. 10 demonstrate that other Shewanella species (e.g., strains EH60:12, EH60:2, and EH60:10) were also capable of releasing oil. Results were comparable to those of strain LH4:18.
Other known Shewanella species were purchased through the German Collection of Microorganisms and Cell Cultures (DSMZ): Shewanella frigidimarina (DSM 12253), S. pacifica (DSM 15445), S. profunda (DSM 15900), S. gelidimarina (DSM 12621), and S. baltica (DSM 9439). Cultures of each strain were grown aerobically overnight in SIB supplemented with 1% peptone. The cultures were then split and 1000 ppm sodium lactate and 2000 ppm sodium nitrate, or 1000 ppm sodium lactate and 3715 ppm sodium fumarate were added. A LOOS test was performed on 2 mL of the cultures as previously described. Samples were not adjusted for growth.
FIG. 11 shows that these known Shewanella species also released oil in the LOOS assay. As in Example 3, those samples grown in the presence of fumarate as the electron acceptor performed better than those grown in the presence of nitrate.
Example 8
Shewanella Increases the Contact Angle of Oil in Deep Sediment Sand
Strain LH4:18 was grown aerobically in PPGAS medium and added to a LOOS test as described above. After approximately two weeks, an aliquot of the sand was removed from the bottom of the strain LH4:18 well and was compared microscopically to oil coated sand from a medium alone control well. FIG. 12 shows photomicrographs for comparison. FIG. 12 A shows the untreated oil coated sand. As indicated by the lines drawn on the picture, the contact angle between the hydrocarbon and sand is low indicating that the surface energy encourages the hydrocarbon to coat the entire mineral grain. The right photomicrograph in FIG. 12B shows the effect of exposure to strain LH4:18. As indicated by the lines drawn on the picture, the contact angle has increased dramatically indicating a significant change in the surface energy between the hydrocarbon and the mineral, and showing substantial liberation of hydrocarbon from the surface of the sand particle. This is a visual demonstration of change in wettability.
Example 9
Measuring Oil Release from Sandpacks
Oil Release Sandpack or Core Flood Assay
The potential application of strain LH4:18 in MEOR treatment was evaluated using the sandpack technique. This was done with an in-house developed Teflon® shrink-wrapped sandpack apparatus. Using a 0.5 inches (1.27 cm) OD and 7 inches (17.78 cm) long Teflon® heat shrink tube, an aluminum inlet fitting with Viton® O-ring was attached to one end of the tube by heat with a heat gun. North Slope sand was added to the column which was vibrated with an engraver to pack down the sand and release trapped air. A second aluminum inlet fitting with Viton® O-ring was attached to the other end of the tube and sealed with heat a gun. The sandpack was then put in an oven at 275° C. for 7 min to evenly heat and shrink the wrap. The sandpack was removed and allowed to cool to room temperature. A second Teflon® heat shrink tube was installed over the original pack and heated in the oven as described above. After the column had cooled, a hose clamp was attached on the pack on the outer wrap over the O-ring and then tightened.
Four sandpack columns were flooded horizontally with three pore volumes of SIB1 low bicarbonate (same as SIB but with 1 mM bicarbonate) at 10 mL/min via a syringe pump and a 60 mL (Becton Dickinson, Franklin Lakes, N.J.) sterile plastic polypropylene syringe. All four sandpacks were then flooded with two pore volumes of anaerobic autoclaved crude oil at 0.5 mL/min to achieve irreducible water saturation. The crude oil was aged on the sand for three weeks before inoculating. For the inoculation culture, strain LH4:18 was grown aerobically overnight in PPGAS medium. The culture was then placed in an anaerobic environment where Na-Lactate was added to 1000 ppm and Na-Nitrate was added to 2000 ppm. This sample was anaerobically aged for 5 days before inoculating the sandpacks. Two columns were anaerobically inoculated with a sample of strain LH4:18 for one pore volume at 0.4 mL/hr. Two control sandpacks were flooded using anaerobic SIB1 low bicarbonate using the same inoculation procedure. The four sandpacks were then shut-in for incubation with the oil for five days. After the shut-in, the columns were produced by flushing with anaerobic sterile SIB low bicarbonate at 0.4 mL/hr for three pore volumes to prepare the production flood.
At the conclusion of the production flood, the 7 inches (17.78 cm) slim tubes were sacrificed into 5 one-inch sections labeled A-E. One inch was skipped at the beginning and at the exit of the slim tube to avoid edge effects during analysis. Sections A, C, and E were analyzed for residual oil saturation on the sand by the GC method described in General Methods.
The results in FIG. 13 show that average residual oil saturation in the uninoculated column was 22.5% whereas the residual oil saturation for strain LH4:18 inoculated columns was 16.1%, indicating that strain LH4:18 was able to reduce residual oil saturation by approximately 6.5%.
Example 10
Discovery of Oil Recovery Activity in Live Injection Water Sample
To screen enrichment cultures, environmental samples or isolated strains for the ability to release oil from a nonporous silica medium, a microtiter plate assay was developed to measure the ability of microbes to release oil/sand from oil-saturated North Slope sand. North Slope sand was autoclaved and then dried under vacuum at 160° C. for 48 hr and 20 g of this dried sand was then mixed with 5 mL of autoclaved, degassed crude oil obtained from Milne point, North Slope. The oil-coated sand was then allowed to adsorb to the sand and age anaerobically at room temperature for at least a week. Microtiter plate assays were set up in a Coy anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). The assay is referred to as the LOOS test (Liberation of Oil Off Sand).
Water samples were obtained from production and injection well heads as mixed oil/water liquids in glass 1.0 L brown bottles, filled to the top, capped and sealed with tape to prevent gas leakage. Gas from inherent anaerobic processes sufficed to maintain anaerobic conditions during shipment. The bottles were shipped in large plastic coolers filled with ice blocks to the testing facilities within 48 hr of sampling.
A sample of non sterile (‘Live’) injection water obtained from Alaskan North Slope was used in a LOOS test plus and minus Shewanella putrefaciens strain LH4:18 (ATCC No. PTA-8822) to determine the efficacy of the Shewanella LH4:18 surface active agent in a background microbial population simulated by the live injection water. Live water was included in the LOOS test as a control. A positive LOOS result was obtained for live injection+/−LH4:18 microbial treatments. The oil/sand release scores obtained from these LOOS tests are given in Table 3.







TABLE 3







Response of Live Injection Water vs. Shewanella LH4:18


in the Release of Oil from Sand in the LOOS Test









Time in days















Live
0
3
7
10
18
26











Test
Injection


Response as diameter of


Sample
Water
LH4:18
Nutrients1
sand released (mm)



















L1
+
−
none
0
1
2
4
4
4


L2
+
−
N
0
1
2
3
3
3


L3
+
−
L/N
0
5
6
6
7
7


L4
+
−
F
0
2
2
4
5
5


L5
+
−
L/N
0
2
3
4
6
6


L6
+
+
none
0
4
4
4
5
5


L7
+
+
N
0
2
4
4
4
4


L8
+
+
L/N
0
4
5
6
6
6


L9
+
+
F
0
4
5
5
6
6


L10
+
+
L/N
0
3
5
6
6
6





1N = Nitrate (2000 ppm); L/N = Lactate (1000 ppm) plus Nitrate (2000 ppm); F = Fumarate (2000 ppm)






The degree of oil release response is measured as the diameter of the sand released from oil. The data demonstrates that test sample L3 consisting of live injection water released oil faster than the other samples. This sample was not inoculated with Shewanella LH4:18. This test demonstrates that the live injection water contained an agent or agents that facilitated the release of oil from sand independent of Shewanella sp LH4:18.
Example 11
Isolation and Identification of Shewanella Species in Oil Reservoir Production Water
Aliquots of the live injection water giving positive oil release results in the LOOS test were streaked on LB agar plates (Teknova, Hollister, Calif.) in order to isolate and identify those strain(s) present in live injection water capable of oil release. Representative colonies with unique morphologies were isolated from the live injection water test samples. Samples of these isolated colonies were screened by PCR amplification using direct colony rDNA analysis described in the General Methods using both the reverse PCR primer 1492r (SEQ ID NO:1) and forward PCR primer 8f (SEQ ID NO:2). The resultant rDNA sequence from each colony was aligned and matched with the GenBank sequence database for phylogenetic strain identification.
One isolate, named L3:3, was identified as having 16S rDNA homology to Shewanella sp C16-M. Both L3:3 and C16-M strains as well as four other reported Shewanella isolates (C31, L31, C13-M and JC5T) have 16S rDNA sequences that are similar to a newly proposed Shewanella species, Sh. chilikensis (K. Sucharita et al, (2009) International Journal of Systematic and Evolutionary Microbiology 59:3111-3115). The 16S rDNA sequence of L3:3 has 99.9% identity to three of the six rDNA gene sequences in the GenBank database that could be classified as Shewanella chilikensis: strain JC5T, strain C16-M, and sequence from a population study designated Shewanella clone D004024H07. Shewanella chilikensis JC5T was isolated from a lake mud environment, Shewanella chilikensis C16-M was isolated from a marine environment and Shewanella clone D004024H07 was isolated (by DuPont) from environmental samples taken from an Alaskan oil well (Pham, V. D, et al., Environ. Microbiol. 11:176-187 (2008)).
Strain L3:3 was identified to be Shewanella sp L3:3 and was further characterized by DNA sequence analysis to have signature sequences within Shewanella species rDNA sequences. Specifically, Shewanella sp L3:3 was found to have 16S rDNA sequence (SEQ ID NO: 3) and signature sequences within Shewanella 16S rRNA variable regions 3 and 6 that are defined in SEQ ID NO:13 (within the prokaryote 16S rRNA variable region 3) and SEQ ID NO: 14 (within the prokaryote 16S rRNA variable region 6). These signature sequence regions were discovered when the 16S rDNA sequence profile of Shewanella sp L3:3 was aligned with 42 published 16S rDNA sequences of Shewanella sp., which were pared down to the nine sequences (SEQ ID NO:4 through 12) in FIGS. 1 and 2 for demonstration of the variations. Shewanella sp L3:3 full 16S rDNA sequence (SEQ ID NO: 3) was used as the alignment anchor. FIG. 14 shows signature base variations that occur in L3:3 in the 16S rRNA variable region 3 and SEQ ID NO: 13 (bp coordinates 430 to 500) at specific coordinate positions: 438-40, 451, 454-57, 466-69, 471, 484-86 and 496 and are observed as signature in nature when compared across 16S rDNA of various Shewanella species. A similar observation was made for bacterial variable region 6 for sequences closely related to Shewanella sp L3:3, e.g., sequences similar to that defined by FIG. 15 and SEQ ID NO: 14 can be found in published sequences. Strain variations occur between base coordinates 990 and 1049, specifically at positions: 995-6, 1001-5, 1007, 1012, 1014, 1016-18 and 1035 as shown in FIG. 2.
In addition to strain L3:3, there are six Shewanella 16S rDNA-like sequences, which were found in sequence databases, that contain the diagnostic signature sequences within variable regions 3 and 6 that are similar to those defined by SEQ ID NO: 13 and SEQ ID NO: 14. This Shewanella group includes: uncultured bacterium clone D004024H07 (NCBI GenBank accession No. gb|EU721813|), Shewanella sp C16-M (gb|EU563338.1|), Shewanella sp. L-10 (gb|DQ164801.1|), Shewanella sp. C31 (gb|EU563345.1|) and Shewanella Sp. JC5T (sp.=chilikensis) (gb|FM210033.2|). Shewanella sp. C13-M (gb|EU563337.1|) does not have the position 471 nucleotide of the L3:3 diagnostic signature.
All strains were isolated from marine environment, oil fields or the bottom of a lagoon. None of these strains at the time of this invention were available from the ATCC or DSMZ public depositories to allow for Ribotyping® comparisons.
Example 12
Riboprint® Analysis of Strain L3:3
To further characterize Shewanella strain L3:3, preparations of this strain were analyzed by Riboprinter® and compared to 7525 patterns contained within DuPont Environmental Services and Qualicon libraries compiled from samples taken all over DuPont as well as another 6950 patterns that DuPont Qualicon has supplied from standard identified organisms. Based on the analyses of Riboprint® batch 052009 (FIG. 16), which provides a chromosomal fingerprint of the tested strains, it is clear that the Riboprint® pattern for strain L3:3 (sample 1) constitutes a Riboprint® which is unique when compared against the available DuPont Riboprint® Libraries and is designated as Ribogroup® identifier 212-824-S-4. It is probable for various strains to share single similar Riboprint® bands generated by hybridizing the labeled E. coli rDNA operon probe to each strain's genomic Eco RI fragments, but it is the overall Riboprint® banding pattern that constitutes identification of a given strain in a specific Riboprint® or Ribogroup® identifier.
Example 13
Enhanced Oil Release by Strain L3:3
The purified strain L3:3 was tested in a LOOS test designed to identify the strains' efficacy in altering the surface tension of oil coated silica particles. Strain L3:3 clearly contributed to oil release from sand as compared to the efficiency of oil release by Shewanella strain LH4:18 (ATCC No. PTA-8822) as shown in Table 4. Both strains exhibited release of oil/sand from oil coated particles. The ability to release oil/sand was similar when fumarate was the electron acceptor for both Shewanella strains tested (LH4:18 and L3:3), but L3:3 appeared to have greater release as compared to LH4:18 when nitrate was used as the electron acceptor.







TABLE 4







LOOS test: Oil/sand Release Response for Purified


Cultures of Shewanella strains L3:3 and LH4:18









Time in Days















0
2
5
7
9
12
14












Electron
Response as diameter of


Test
Strain
receptor
sand released (mm)



















1
L3:3
none
0
4
6
7
7
8
8


2
L3:3
L/N1
0
2
5
5
7
7
8


3
L3:3
L/F2
0
3
7
7
7
8
8


4
LH4:18
none
0
2
6
7
7
7
7


5
LH4:18
L/N
0
0
3
3
3
4
4


L6
LH4:18
L/F
0
3
6
7
7
7
7





1lactate plus nitrate


2lactate plus fumarate






Example 14 (Prophetic)
Anaerobic Growth of Strain L3:3 on Oil as the Sole Carbon Source
To study growth of strain L3:3 as compared to Shewanella LH4:18, purified isolates are inoculated into 20 mL serum vials containing  ̃10 mL minimal salts medium (Table 5), 1.6 g/l sodium nitrate and 5.0 mL of autoclaved crude oil. The medium is deoxygenated by sparging the filled vials with a mixture of nitrogen and carbon dioxide followed by autoclaving. All manipulations of bacteria are done in an anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). The cultures are incubated at ambient temperatures with moderate shaking (100 rpm) for several weeks to several months and monitored for nitrate, nitrite, visible turbidity and visible oil modifications. When the nitrate is depleted in any culture, sodium nitrate at 50 g/l is added to bring its concentration in the medium up to 0.4 g/l sodium nitrate.







TABLE 5







Minimal salts medium










Final
Chemical


Growth component
concentration
Source













Nitrogen
18.7
μM
NH4Cl


Phosphorus
3.7
μM
KH2PO4


Magnesium
984
μM
MgCl2•6H2O


Calcium
680
μM
CaCL2•2H2O


Sodium chloride
172
mM
NaCl


Trace metals






670
μM
nitrilotriacetic acid



15.1
μM
FeCl2•4H2O



1.2
μM
CuCl2•2H2O



5.1
μM
MnCL2•4H2O



12.6
μM
CoCl2•6H2O



7.3
μM
ZnCl2



1.6
μM
H3BO3



0.4
μM
Na2MoO4•2H2O



7.6
μM
NiCl2•6H2O


pH buffer (7.5 final)
10
mM
Hepes


Selenium-tungstate
22.8
nM
Na2SeO3•5H2O



24.3
nM
Na2WO4•2H2O


Bicarbonate
23.8
nM
NaHCO3


vitamins
100
μg/L
vitamin B12



80
μg/L
p-aminobenzoic acid



20
μg/L
nicotinic acid



100
μg/L
calcium pantothenate



300
μg/L
pyridoxine hydrochloride



200
μg/L
thiamine-HCl•2H2O



50
μg/L
alpha-lipoic acid


Electron acceptor
1.0.
g/L
NaNO3, Na2 fumarate or





Fe(III) Na EDTA





The pH of the medium is adjusted to 7.5.






Strain L3:3 is expected to show growth via nitrate reduction and turbidity increase under denitrifying conditions as does LH4:18.

Example 15
Anaerobic Growth of Strain L3:3 on Oil as the Sole Carbon Source
Strain L3:3 and Shewanella strain LH4:18 were studied and compared in their abilities for anaerobic growth on oil as the sole carbon source using different electron acceptors. Shewanella strain LH4:18 has been show to grow using nitrate as the electron acceptor in commonly owned and co-pending US 2009-0260803 A1. Shewanella strains L3:3 and LH4:18 were inoculated into 20 mL serum vials containing  ̃10 mL SL10 minimal salts medium (Table 2), supplemented with one of the following electron acceptors: NaNO3 (2000 ppm), Na2 fumarate (3500 ppm), or Fe(III) Na EDTA (5000 ppm), and overlayed with 5.0 mL of autoclaved crude oil. LH4-18 samples were excluded from NaNO3 test vials. The medium and crude oil had been deoxygenated by sparging these reagents with a mixture of carbon dioxide and nitrogen (20% and 80%, respectively) followed by autoclaving. All manipulations of bacteria were done in an anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.) (gas mixture: 5% hydrogen, 10% carbon dioxide and 85% nitrogen). Replicate test vials were set up per electron acceptor treatment by L3-3 inoculum. The cultures were incubated at ambient temperature for two weeks. Cell growth/titer of the test cultures were analyzed by MPNs.
L3:3 grew anaerobically in oil enrichments where crude oil was provided as the sole carbon source when either NaNO3, Na2 fumarate, or Fe (III) Na EDTA was provided as the electron acceptor. A table of growth results as analyzed by cell titers recorded as MPN log 10 is listed below in Table. 6. Strain L3:3 grew anaerobically 3 logs to cell titers of  ̃105-107 cells per mL from starting titers of  ̃103 cells per mL after two weeks incubation time. The change in cell numbers as a result of anaerobic growth on oil are listed as the log10 of the MPN recorded for growth±0.5 log. The growth of Shewanella strain L3:3 on the different electron acceptors was comparable to that of Shewanella strain LH4:18. Strain L3:3 grew anaerobically on oil using either NaNO3, Na2 fumarate, or Fe (III) Na EDTA as the electron acceptor. Cell titers are presented as the average of replicate test vials. Shewanella strain LH4:18 also grew on oil using fumarate or Fe (III) Na EDTA as electron acceptor. Its growth on oil using nitrate as an electron acceptor had been previously demonstrated in commonly owned and co-pending US 2009-0260803 A1. Both L3:3 and Shewanella strain LH4:18 grew  ̃3 logs to titers of  ̃105-107 cells per mL from starting titers of 103 cells per mL anaerobically after two weeks incubation time.







TABLE 6







Anaerobic Growth on oil









Delta MPN log10 Electron Acceptor










Strain
NaNO3
Na2 fumarate
Fe(III) Na EDTA





Shewanella strain L3:3
3.0
3.2
2.7


Shewanella strain LH4:18
n.t.
4.7
2.2





n.t. = not tested






Example 16
Determining the Temperature Limits for Growth of Strain L3:3
To determine the optimal temperature range as well as tolerances for Shewanella putrefaciens LH4:18 and Shewanella sp L3:3, these strains were grown at different test temperatures as given in Table 7.
Inoculums of Shewanella strains LH4:18 and L3:3 were grown overnight (16 to 18 hours) aerobically in LB medium with shaking at 30-35° C. These overnight cultures were grown to visible turbidity and relatively high levels of cell counts as measured by optical density. Aliquots from these starter cultures were then used to seed flasks of 10 mL sterile LB media that had been pre-incubated at a specific test temperature over night. Temperature test cultures were seeded at an optical density of approximately 0.1 as measured using a spectrophotometer and visible light, wavelength of 600 nm. This constituted a dilution of starter cultures approximating between a 1:50 and 1:100 dilution. Growth was then measured by tracking the turbidity or optical density of cultures over time. The resulting growth rates determined for strains LH4:18 and L3:3 obtained for the different test temperatures is expressed as doubling time, the time to double cell number, in units of hours; the smaller the number the faster the growth rate. At doubling times of <2 h these strains are presumed to compete successfully with background microbial populations in situ. Table 7 shows the results for growth rates at the recorded temperatures. Both Shewanella strains were shown to grow at a rate that would allow them to compete with microbial populations in situ for a relatively broad range of environmental temperatures.







TABLE 7







Average Recorded Doubling Time for selected temperatures ° C.









Doubling times (in hours)



Temperature
















strain
10° C.
16° C.
22° C.
27° C.
30° C.
32° C.
35° C.
37° C.
41° C.





LH4:18
3.18
1.71
1.02
0.95
0.87
0.79*
0.97
1.45
No growth


L3:3
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.98
0.63





.n.t. = not tested






Example 17
Anaerobic Growth of Strain L3:3 in the Presence of Oil Under Denitrifying Conditions
To demonstrate the ability to grow anaerobically in the presence of oil under denitrifying conditions, an aliquot (104-105 cells) of each of Shewanella strains LH4:18 and L3:3 was inoculated under anaerobic conditions into 20 mL serum vials containing a 1:2 ratio of minimal salts medium supplemented with sodium lactate. The media formulation used was designed to promote growth and propagation of Shewanella strain L3:3 as well as the oil release mechanism within a reservoir environment. The medium composition for anaerobic growth was as follows: 10 mL minimal salts medium (Table 4 minimal salts medium), 1000 ppm sodium lactate and  ̃2000 ppm sodium nitrate with 5.0 mL of autoclaved crude oil. Strain LH4:18 acted as a positive control for anaerobic growth under denitrifying conditions containing surface active agent(s). Both the medium and crude oil were deoxygenated by sparging the filled vials with a mixture of nitrogen and carbon dioxide followed by autoclaving. All manipulations of bacteria were done in an anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). These cultures were incubated at ambient temperatures for several days and monitored for nitrate and nitrite levels, for visible turbidity, and for visible changes to the integrity of the oil phase.
Table 8 shows the results of this growth study in the presence of oil. A pure culture of strain L3:3 showed growth through a reduction in lactate and nitrate levels when grown in the presence of oil. These strains also showed  ̃two to three logs growth as indicated by MPN data (Table 8).







TABLE 8







Nitrate Reduction as a Measure of Anaerobic Growth In the


presence of Oil with lactate as the primary carbon source














% nitrate
% Lactate




Bacteria
16S Genus
reduction
reduction
MPN
time


isolate
ID
in oil
in oil
log10
(days)





NIC1
n.a.
 8%
 0%
n.t.2
14


L3:3
Shewanella
58%
44%
7.0
14



chilikensis,







JC5T






LH4:18
Shewanella
51%
35%
6.4
14



putrefaciens





1NIC: Non inoculated control


2n.t.: not tested






Example 18
Identification of Electron Acceptors which Promote Oil Release by Strain L3:3
Different terminal electron acceptors (shown in Table 9) were tested in anaerobic growth of strain L3:3 to determine its ability to grow on a range of terminal electron acceptors including fumarate as well as various metal oxides. A mixed culture of LH4:18 and L3:3 was also tested with nitrate and fumarate. Anaerobic test growths were set up using minimal salts media (Table 4). 20 mL of minimal salts medium was supplemented with 1000 ppm sodium lactate, where 2000 ppm sodium nitrate was used as the electron acceptor control. The milli-equivalents of the following electron acceptors were each applied in their respective electron assay sample: fumarate, pyruvate, Fe (III) sodium EDTA, manganese dioxide, and vanadium dioxide. The minimal salts base medium, lactate, and terminal electron acceptor preparations were all deoxygenated by sparging with a mixture of nitrogen and carbon dioxide followed by autoclaving. All manipulations of bacteria were done in an anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). These cultures were incubated at ambient temperature for several days and monitored for growth by increases in visible turbidity as measured by OD/MPN or by lactate depletion as measured by IC. Results are shown in Table 9.







TABLE 9







Relative Growth obtained for Strains L3:3 and LH4:18 on different


electron acceptors using Lactate as supplemental Carbon Source
















Iron






Ni-
Fuma-
(Fe(III))
Manganese
Vanadium
Pyru-



trate
rate
EDTA
Dioxide
Dioxide
vate

















L3:3
+
++
++
++
+
++


L3:3 +
+
++
n.t.1
n.t.
n.t.
n.t.


LH4:18





1n.t. not tested






Example 19
Strain L3:3 Increases the Contact Angle of Oil on Deep Sediment Sand
Strain L3:3 was grown aerobically overnight in SIB (Synthetic Injection Brine; Table 10) plus 1% peptone. Samples were then added into an anaerobic LOOS test, described above, and were supplemented with 1000 ppm sodium lactate and 2000 ppm sodium nitrate. After approximately one week, an aliquot of the sand was removed from the bottom of the strain L3:3 well and was visualized microscopically. FIG. 17(A) is a typical image of untreated oil coated sand. As indicated, the contact angle (qCA) between the hydrocarbon and sand is low—the surface energy encourages the hydrocarbon to coat the entire mineral grain. FIG. 17(B) shows the effect of exposure of oil coated sand to strain L3:3. The contact angle (qCB) is increased dramatically indicating a significant change in the surface energy between the hydrocarbon and the mineral







TABLE 10





Components of SIB1 Minimal Medium (per Liter)


and added electron acceptor and electron donor



















NaHCO3
0.138
g



CaCl2*6H2O
0.39
g



MgCl2*6H2O
0.220
g



KCl
0.090
g



NaCl
11.60
g



Trace metals (Table 4)
1
ml



Vitamins (Table 4)
1
ml










Na2HPO4
0.015 g (10 ppm PO4)



NH4Cl
0.029 g (10 ppm NH4)







Electron donor added










Na-Lactate
0.124 g (124 ppm Na-




Lactate)







Electron acceptor added










Na2nitrate
0.4 g/400 ppm





Adjust pH with HCl or NaOH


Filter sterilize






Example 20
Demonstration of Strain L3:3 Oil Release Sandpack or Core Flood Assay
To test the amount of residual oil left in a sandpack after the oil soaked sandpack was flooded with a water solution that simulated the injection brine used in flooding an underground oil reservoir, the sandpack was fabricated as per standard methods described by Petroleum Reservoir Rock and Fluid Properties, Abhijit Y. Dandehar, CRC Press (2006). A similar core flood/sandpack apparatus and techniques used to operate it are also described by Berry et al. (SPE paper number 200056, SPE Reservoir Engineering, November 1991, p 429). The use of a similar apparatus and techniques for testing microbial treatments in a sandpack is described by Saikrishna et al. (SPE paper number 89473, (2004)).
To demonstrate that strain L3:3 is capable of oil release, a L3:3 culture was applied to a sandpack saturated with oil in an in-house developed Teflon® shrink-wrapped sandpack apparatus that simulates packed sand of sandstone. The process described herein was used for making two column sets, a “control” set and a “test” set, which was inoculated with L3:3 to test its efficacy to release oil from the sand column. Using a 1.1 inches (2.8 cm) diameter, and 7 inches (17.8 cm) long Teflon heat shrink tube, an aluminum inlet fitting with Viton® O-ring was attached to one end of the tube using a heat gun. Alaskan North Slope sand was added to the column which was vibrated with an engraver to pack down the sand and release trapped air. A second aluminum inlet fitting with Viton® O-ring was attached to the other end of the tube and sealed with heat a gun. The sandpack was then put in an oven at 275° C. for 7 min to evenly heat and shrink the wrap. The sandpack was removed and allowed to cool to room temperature. A second Teflon® heat shrink tube was installed over the original pack and heated in the oven as described above. After the column had cooled, a hose clamp was attached on the pack on the outer wrap over the O-ring and then tightened. For this demonstration there were four sandpack columns assembled.
The four sandpack columns were flooded horizontally with three pore volumes of SIB1 Synthetic Injection Brine (Table 10) at 10 mL/min via a syringe pump and a 60 mL (BD) sterile plastic polypropylene syringe. All four sandpacks were then flooded with two pore volumes of anaerobic autoclaved crude oil at 0.5 mL/min to achieve irreducible water saturation. The crude oil was aged on the sand for three weeks prior to being inoculated with strain L3:3.
For inoculation, the culture was grown aerobically overnight in PPGAS media (Table 11). The culture was then placed in an anaerobic environment where sodium lactate was added to SIB1 minimal brine solution to a concentration of 1000 ppm and sodium nitrate was added to a concentration of 2000 ppm. The inoculation sample was then anaerobically aged in an anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.) for 5 days before inoculating the sandpacks. After the aging period, two columns were anaerobically inoculated with a sample of Shewanella sp L3:3 for one pore volume at 0.4 mL/hr. Two control sandpacks were flooded using anaerobic SIB1, using the same inoculation procedure. The four sandpacks were then shut-in for incubation with the oil for five days. After the shut-in, the columns were then produced for three pore volumes with anaerobic sterile SIB1 low bicarbonate at 0.4 mL/hr.







TABLE 11





Components for PPGAS Growth Medium (per Liter)



















Peptone
10
g



Mg SO4
0.2
g



KCl
1.5
g



NH4 CL
1.07
g



Tris HCL Buffer, pH 7.5
120
mL









At the conclusion of the production flood, the 7 inches long slim tubes were sacrificed into three 1.9-inch sections labeled A-C. One inch was skipped at the beginning and at the exit of the slim tube to avoid edge effects during analysis. Section “A” came from the front end of the column. Sections A, B and C were analyzed for residual oil saturation on the sand. The amount of oil on the wet sand from the sacrificed slim tubes for residual oil was measured by GC as described above. This value was multiplied by the total amount of toluene used to extract the oil resulting in the total amount of oil on the sand. The value obtained was then divided by the total sample weight to yield the percent of oil with respect to the total sample weight. The weight percent of oil of the sample was then multiplied by the ratio of the empirically derived characteristic of packed North Slope sand (total weight of sample after being flooded with brine divided by total sand weight, 1.27). This relationship is equal to the amount of oil on dry sand. This value was then multiplied by the ratio of the weight of the North Slope sand to the weight of the fluid trapped in the pore space of the sand, 3.75. The resulting value reflected the residual oil left on the sand in units of g of oil/g of total fluid in the pore space. As shown in Table 12, residual oil left on the column, in fractions A and C of the test column, were less than the controls confirming that the columns inoculated with the Shewanella sp. L3:3 released more oil than uninoculated control columns, with an average of 4.1% decrease in residual oil remaining on the columns when L3:3 was inoculated on the columns.







TABLE 12







Residual oil left on sand along the tube length after


flooding with anaerobic sterile “Brine”









Average Percent Residual Oil on Sand










Column Fraction













Assay Column
A
C
Average.






Test columns
12.6%
15.3%
13.9%



Control columns
18.9%
17.1%
18.0%









Example 21
Isolation of Strain MPHPW-1
Oil well production water collected from an oil reservoir near Wainwright, Alberta Canada was enriched by supplementing with 1% Bacto Peptone (Becton Dickinson, Franklin Lakes, N.J.) and cultured by incubating aerobically overnight at room temperature (21-24° C.) with shaking at 220 rpm. This culture was further enriched during an anaerobic LOOS oil release assay that was run as described in General Methods. An aliquot of the overnight culture received 1000 ppm sodium lactate and 2000 ppm sodium nitrate. Samples were then run in the anaerobic oil release test for approximately two weeks. Samples from microtiter plate wells in which oil was released were then plated onto LB agar plates (Teknova, Hollister, Calif., USA) to obtain pure isolates. Representative colonies with unique morphologies were streaked again onto LB plates and one strain was selected for further testing, which was named MPHPW-1.
Example 22
Demonstration of Oil Release Potential of Strain MPHPW-1
To further assess the ability of strain MPHPW-1 to release oil from oil saturated sand, an oil release test was performed. Briefly, a small volume of MPHPW-1 frozen stock was plated onto an LB agar plate and allowed to grow at room temperature. Simulated injection brine plus 1% peptone was inoculated with an individual colony from the plate. The culture was incubated aerobically overnight at room temperature (21-24° C.) with shaking at 220 rpm and grown to a cell density of approximately 1×109 CFU/ml, as determined using the MPN assay described in General Methods. An anaerobic LOOS test was performed as described in General Methods. A control sample contained medium (simulated injection brine plus 1% peptone) alone. Sand/oil release was compared in the MPHPW-1 and control samples over time. The oil release results were that strain MPHPW-1 released oil from a sample of oil soaked sand while the medium alone control released no oil (FIG. 19).
Example 23
Demonstration of Oil Release by Strain MPHPW-1 Grown in the Presence of Different Nutrients
Strain MPHPW-1 was grown in the presence of different media supplements. MPHPW-1 was inoculated into SHS-10 medium (171 mM NaCl, 0.98 mM MgCl2, 1.4 mM CaCl2, 0.1 mM KCl, 0.16 mM Na2SO4, 16.4 mM NaHCO3) supplemented with 1% of Amberferm 2382 (Sensient Flavors, Inc., Harbor Beach, Mich.), Amberferm 2391 (Sensient Flavors, Inc), Tastone (Sensient Flavors, Inc.), or Bacto peptone. These supplements are different sources of small peptides and free amino acids. The cultures were incubated aerobically overnight at room temperature (21-24° C.) with shaking at 220 rpm. An anaerobic LOOS test was performed as described in General Methods. Sand/oil release was compared across all samples over time. The results in FIG. 20 showed that strain MPHPW-1 released oil when grown in the presence of the different supplements.
Example 24
Measuring Oil Release by MPHPW-1 from Sandpacks
The potential application of strain MPHPW-1 in MEOR treatment was evaluated using a sandpack assay. This was done with an in-house sandpack. A schematic diagram of the sandpack experimental set-up is shown in FIG. 21. All numbers below in bold refer to FIG. 21.
A sample of produced sand that was obtained from the Schrader Bluff formation at the Milne Point Unit of the Alaska North Slope was cleaned by washing with a solvent made up of a 50/50 (volume/volume) mixture of methanol and toluene. The solvent was subsequently drained and then evaporated off the sand to produce clean, dry, flowable sand. This sand was sieved to remove particles less than one micrometer in size. The sand was packed tightly into a 20 cm long and 3 cm inner diameter, flexible heat shrinkable tubing (23) and compacted by vibration using a laboratory engraver.
Both ends of the sandpack were capped with compression type fittings to retain the sand mix. Flexible 1⁄8 inch (0.32 cm) tubing capable of sustaining the pressures used in the test was attached to the fittings. The sandpack was mounted into a pressure vessel (19) with the tubing (18 and 24) passing through the ends of the pressure vessel using commonly available bulkhead pressure fittings (20). Additional fittings and tubing were used to connect the inlet of the sandpack to a pressure pump (16), and an injection brine reservoir (15). Low flow rate of a concentrated solution of nutrients could be pumped using a common syringe pump (17) and diluted into the brine being fed from the brine reservoir (15). Other common compression fittings, including elbow unions and tees, and tubing connected the inlet of the sandpack to a transducer that measured the pressure above atmospheric pressure (absolute pressure gauge) (26). The inlet of the sandpack was also connected using the same types of tubing and fittings to the high pressure side of a commonly available differential pressure transducer (27). Fittings and tubing connected the outlet of the sandpack to the low pressure side of the differential pressure transducer (27) and to a back pressure regulator (28). The produced fluid was collected in a jug (29) and periodically weighed using a weigh scale (30) in order to confirm the flow rate of the feed pump (16). The pressure vessel (19), the sandpack (23) and the bulkhead fittings (20) were weighed as well, as a means to help determine the amount of oil left in the sandpack. The signals from the weigh scales (25, 30), and the differential pressure and the absolute pressure transducers (26, 27) were ported to a computer and these signals were monitored and periodically recorded. The pressure vessel (19) around the sandpack was pressurized using nitrogen ported into the pressure vessel through flexible tubing (22). The nitrogen pressure was monitored using a pressure gauge (21). The nitrogen was at a pressure of about 110 pounds per square inch (psi) (0.74 mega Pascal) while Brine from the feed reservoir (15) flowed through the sandpack and came out through the back pressure regulator (28). This operation was performed such that the pressure in the sandpack (23) was always 5 to 20 psi (0.034-0.137 mega Pascal) below the pressure in the pressure vessel (19).
Throughout the following experimental protocol, the sandpack was operated under pressure between 85 to 90 psig so any gas would remain dissolved, thus avoiding air or gas occupying the void volume. Before beginning the experiment, the sandpack was conditioned by flowing brine through the pack at 4 mL/hour for more than two months.
The sandpack was flooded vertically with three pore volumes of sterile simulated injection brine containing 40 ppt NaCl at 4 mL/min. The sandpack was then flooded with two pore volumes of anaerobic autoclaved crude oil at 4.0 mL/min to achieve irreducible water saturation. The crude oil was aged on the sand for one week and then flooded off using the same brine as above. This process was repeated three more times to insure that all hysteresis had been removed from the system and that a reproducible level of oil saturation could be regenerated with each oiling and de-oiling of the system.
When this was accomplished the system was ready for inoculation. For the inoculation culture one pore volume of strain MPHPW-1 was grown aerobically overnight in SHS-10 medium (171 mM NaCl, 0.98 mM MgCl2, 1.4 mM CaCl2, 0.1 mM KCl, 0.16 mM Na2SO4, 16.4 mM NaHCO3) plus 1% Amberferm 2391. Just before inoculation of the sandpack, 1000 ppm Na-Lactate and 3715 ppm Na-Fumarate were added to the inoculum. The sandpack column was inoculated with this MPHPW-1 culture at 4.0 mL/min and was shut-in for 5 days. After the shut-in, the column was produced by flushing using the simulated injection brine with 40 ppt NaCl at 4.0 mL/hr for three pore volumes.
The percent of volume occupied by water, also called water saturation, was calculated during the experiment from the monitored weight of the sandpack. The water saturation calculation was performed as follows: The difference in oil and water densities along with the void volume of the sandpack together determine the difference in weight of the sandpack if the void volume were all oil or all water. The measured density of the oil used in the experiment was 0.91 g/ml and the density of water is 1.0 g/ml.
Water Saturation Calculations Used for FIG. 22:
First the theoretical weight swing on column from 100% water to 100% oil was calculated as follows:
30% void fraction (estimated)
0.09 g/cc density difference (1 g/cc water, 0.91 g/cc oil)
116 total volume of sandpack in cc (measured  ̃1.1 inches in diameter×7.4 to 7.5 inches in length; used 1.1 inches diameter, 7.45 inches length for calculation)
3.1 theoretical weight swing from 100% oil to 100% water
0.30×(0.09×116)=3.1 g. (theoretical weight swing)
Oil density was measured; water density is known in the art; 30% void fraction is known in the art.
Next, Column was flooded with oil to the point at which the weight of the apparatus did not change. This corresponded to its irreducible water saturation; the column weight was measured as −2.702 grams and this weight was used in subsequent calculations as corresponding to 19% water content.
The art and prior work with these types of sand packs has shown us that the irreducible water saturation or residual water saturation will be around 10 to 30%. We chose 19% to use in these calculations. It is not the absolute value that is important; it is the change in the water saturation units or % void volume that is important.
Next, the amount of oil on column after SIB treatment was determined (baseline to determine effectiveness of subsequent medium-alone and MPHPW-1 treatments).

Calculation:

(measured weight after oil-saturated column washed with SIB)−(Weight with 100% SIB)=−1.134 g.

Increased water saturation=(−1.134−(−2.702))/3.1×100%=50% increase in water saturation.

Since the starting water saturation was 19%, then the new water saturation is 50+19=69%.

The amount of oil on column after medium treatment was determined, and then medium plus MPHPW-1 (referenced in calculation as test solution, meant to denote separate washes, first with just medium, and then with MPHPW-1 plus medium).

Calculation:

(observed weight of column−(−2.702)/3.1×100%+19%=the water saturation units plotted in FIG. 22.

This calculated water saturation was graphed in FIG. 22, with the Y axis, labeled “% void volume occupied by water” corresponding to % water saturation, and the X axis corresponding to the number of void volumes flooded by each solution. The water saturation percentage is a measurement of effectiveness of oil removal or recovery.
The results showed that MPHPW-1 treatment increased the average water saturation by about 15%. In FIG. 22, the first part of the curve, labeled 1, is the deoiling curve using injection brine alone. It shows that after the column has been saturated with oil and flushed with water, the water saturation is 69%. The second part of the curve, labeled 2 is the deoiling curve using medium alone without any microorganisms added. There appears to be an increased water saturation of about 2% (5) The third part of the curve, labeled 3 is the deoiling curve using medium plus MPHPW-1. There appears to be an increase in water saturation of about 15% (4).
Example 25
LOOS Test Confirmation of the Oil Release of the Sandpack Inoculum
To confirm that the MPHPW-1 inoculum used in the sandpack (Example 24 above) released oil from oil saturated sand, a standard LOOS test was performed as described in General Methods using SHS-10 plus 1% Amberferm medium. The effect on oil release of a sample of the inoculum used in Example 24 was compared to the effect of SHS-10 plus 1% Amberferm medium alone. Results are shown in FIG. 23. The MPHPW-1 sandpack inoculum released oil whereas the medium alone did not in the first 12 days of the assay.
Example 26
Identification of Strain MPHPW-1 as a New Strain of Shewanella 
Strain MPHPW-1 was identified as a strain of Shewanella, most closely related to S. algae strain BrY, by using 16S rDNA sequence analysis consistent with the criteria set forth in the International Journal of Systematic and Evolutionary Microbiology (B. J. Tindall, R. Rosselló-Mora, H.-J. Busse, W. Ludwig and P. Kämpfer, Int. J. Syst. Evol. Microbiol. (2010), 60:249-266).
Genomic DNA was isolated from a pure single colony of this strain that was isolated as described in Example 21. The universal primers Reverse Primer 1492R (SEQ ID NO:1) and Forward Primer 8F (SEQ ID NO:2) were used to PCR-amplify a near full length 16S rDNA genomic DNA fragment of about 1450 bp from this isolate. This amplified fragment was cloned and then sequenced five times in each direction and the raw data was compiled to obtain the final sequence (SEQ ID NO: 24). The resulting consensus sequence (SEQ ID NO:24) was queried against the NCBI (The National Center for Biotechnology Information) nucleic acid database, using the BLAST (Basic Local Alignment Search Tool) algorithm. High identity sequence hits were sequences of 16S rDNA from strains of Shewanella species. There were no sequences found having 100% identity to SEQ ID NO:24.
The 16S rDNA sequences of the type strains of the 55 recognized species of Shewanella from the List of Prokaryotic names with Standing in Nomenclature (LPNSN), as well as a few additional representative strains that are listed in Table 13 were downloaded from the NCBI Genbank nucleic acid database. A multiple sequence alignment was performed anchored by the E. coli K12 16S rDNA B sequence, which is recognized as the standard 16S rDNA for base position assignment (Brosius, Jürge, et al., 1981, J. Mol. Biology. 148:107; Woese, C. R. 1987. Bacterial Evolution. Microbial. Rev. 51:221), to provide base coordinate positions. The alignment was performed using the global multiple sequence alignment algorithm from the Clustal series of programs, Clustal W, DNAstar Lasergene Version 8.0.3 MegAlign package, Madison Wis. (Chenna, Ramu, et al., (2003) Nucl. Acids Res. 31:3497). Sequences were aligned across their entire length. All sequences used in the alignment were from near full length 16S rDNA sequence, which starts at E. coli coordinate base No. 61 and ends at base coordinate 1460. MPHPW-1 coordinates in SEQ ID NO:24 that correspond to the E. coli coordinates 61-1460 are MPHPW-1 base coordinates 17 (5′-GTCGA) through 1418 (GGGC-3′). Four exceptions with regard to sequence coverage from E. coli coordinates 61 to 1460 were the DNA sequences from the following type strains, which were not complete at the 5′ or 3′ termini
Shewanella atlantica strain HAW-EB5 (CCUG 54554; Genbank:AY579752, SEQ ID:71) sequence from positions 83 to 1376;
Shewanella canadensis strain HAW-EB2 (CCUG 54553:Genbank AY579749, SEQ ID:68) sequence from positions 83 to 1368;
Shewanella sediminis strain HAW-EB3 (DSM 17055: Genbank CP000821, SEQ ID:89) sequence from positions 83 to 1363;
Shewanella profunda strain LT13a (DSM 15900:Genbank AY445591, SEQ ID:64) sequence from positions 198 to 1531.
The multiple sequence alignment showed that among all pairs of the aligned Shewanella type species sequences, and representative species sequences that were included, there was at least 90% identity with MPHPW-1 confirming that MPHPW-1 is a strain of Shewanella. 
The Guide Tree, which resembles a phylogenetic tree, in FIG. 24 is based on a sequence distance method and utilizes the Neighbor Joining (NJ) algorithm of Saitou and Nei (Saitou, N. and Nei, M. (1987), The neighbor-joining method: a new method for reconstructing Guide Trees. Mol. Biol. Evol. 4; 406-425). The NJ method works on a matrix of distances between all pairs of sequence to be analyzed. These distances are related to the degree of divergence between the sequences. The Guide Tree is calculated after the sequences are aligned. AlignX displays the calculated distance values in parenthesis following the molecule name displayed on the tree for genus Shewanella strains and strain MPHPW-1 based on similarity and differences in 16S rRNA gene sequences was generated from this alignment using bootstrap and Clustal W analysis (Vector NTI AlignX software package 10.3.1 Invitrogen, Carlsbad, Calif.). The software positioned strain MPHPW-1 in a phylogenetic clade consisting of four species: Shewanella algae, Shewanella haliotis, Shewanella chilikensis and Shewanella marinus. Shewanella algae strain BrY and strain MPHPW-1 formed a sub-clade within this clade that was separate from the other Shewanella algae species of the clade, represented by type strain Shewanella algae OK-1 (ATCC 51192). Based on the close relationship of MPHPW-1 and Shewanella. algae BrY, MPHPW-1 was determined to belong to the Shewanella algae species. Shewanella sp. strain KJW27 (16S rDNA Accession #HM016084) was also most closely related to S. algae BrY by 16S rDNA sequence analysis and has 2 position differences with the MPHPW-1 16S rDNA. The Shewanella sp. KJW27 strain is slated to be named a new species, Shewanella indica, by the International Journal of Systematic and Evolutionary Microbiology (personal communication). The naming of this new species and the relationships that it has with BrY and MPHPW-1 may indicate the assignment of MPHPW-1 as a Shewanella indica once the new naming is published.
By analyzing the ClustalW alignment, signature sequence positions in the 16S rDNA sequence were identified that could be used to distinguish different Shewanella species and also genomavars within the Shewanella algae species. These signature positions are listed in Table 14 with position coordinate numbers of the E. coli K12 W3110 rrnB allele for 16S rDNA sequence. Table 14 lists the hypervariable region of 16S RNA sequence, where each listed signature sequence position is located. Approximate positions of the hypervariable regions designated by nucleotides of the 16S rDNA sequence from E. coli are:
hypervariable region 1 between positions 60 and 99;
hypervariable region 2 between positions 118 and 290;
hypervariable region 3 between positions 410 and 520;
hypervariable region 4 between positions 578 and 760;
hypervariable region 5 between positions 820 and 888;
hypervariable region 6 between positions 980 and 1048;
hypervariable region 7 between positions 1071 and 1179;
hypervariable region 8 between positions 1215 and 1335;
hypervariable region 9 between positions 1350 and 1480.
In Table 14, the sequences at the signature positions are given for Shewanella algae strains ATCC51192 (16S rDNA SEQ ID NO:45), FeRed (16S rDNA SEQ ID NO:90), and BrY (16S rDNA SEQ ID NO:27), and the closest strains on the phylogenetic tree (with >98% sequence identity): Shewanella haliotis (16S rDNA SEQ ID NO:33), and Shewanella chilikensis (16S rDNA SEQ ID NO:10). All of these strains have the same signature sequence at the positions shown in the boxed regions of the table, for example in hypervariable region 1 at positions 74-84 and 88-98. The signature sequences for less closely related strains Shewanella oneidensis (16S rDNA SEQ ID NO:86), Shewanella colwelliana (16S rDNA SEQ ID NO:73), and Shewanella amazonensis (16S rDNA SEQ ID NO:44), also shown in this table, differ at these positions.
A set of signature sequences that distinguished Shewanella algae BrY and strain MPHPW-1 from the other Shewanella strains occurred at 15 specific positions: two in variable region 2 (positions 264 and 278), two in variable region 3 (positions 456-463, 488-491), four in variable region 5 (positions 847, 853, 856 and 858), four in variable region 6 (positions 1000-1001, 1006-1012, 1017-1023 and 1039-1040) and three in variable region 8 (positions 1243-1245, 1283 and 1292-1294). The Shewanella algae BrY sequence differs from that of MPHPW-1 as follows: mismatches at positions 163 and 170 in the MPHPW-1 sequence (positions 203 and 213 using E. coli 16S rDNA position numbers as in Table 14 below), and deletions from MPHPW-1 following positions 16, 37, and 1421 of the MPHPW-1 sequence.
The sequences with closest identity to the MPHPW-1 16S rDNA sequence from the BLAST search, other than S. algae BrY, are listed in Table 15. Due to the sequence identities with other Shewanella strains, one of these sequences appear to be misclassified as belonging to a strain of Rhodobacter capsulatus rather than Shewanella. Each of these sequences had at least four position differences with the sequence of MPHPW-1, including nucleotide changes, insertions, and deletions. Thus, based on the 16S rDNA sequence analysis, MPHPW-1 was identified as a new strain of Shewanella algae. 
The 16S rDNA sequence of MPHPW-1 fell within the Shewanella degenerate signature sequences shown in FIG. 18 with an exception at position 23 of SEQ ID NO:19. This position is number 199 with respect to the E. coli 16S rDNA positioning standard. In the MPHPW-1 sequence there is a C at this position. Thus to incorporate the MPHPW-1 sequence at this position into the Shewanella degenerate sequence, the degenerate designation was changed from R (which includes A or G) to V (which includes A, C, or G). The revised Shewanella degenerate signature sequence for the 16S variable region 2 is SEQ ID NO:25.
The Shewanella degenerate signature sequence for variable region 2 that specifies MPHPW-1 and the other members of the clade to which it belongs (Shewanella algae, Shewanella haliotis, Shewanella chilikensis and Shewanella marinus) contains all of the degeneracy in SEQ ID NO:25 except C is specified at position 23 of region 2 (SEQ ID NO:28).







TABLE 13







Type strains and representative strains of Shewanella species, with GenBank


accession numbers to reference rRNA encoding gene sequences used in alignment












Strain name and
SEQ


Genus and Species
Accession No.
Deposit Identification
ID NO





Shewanella putrefaciens
X81623
Hammer 95 = ATCC
32




8071.



Shewanella hanedai
X82132
281 = ATCC 33224
31


Shewanella benthica
X82131
W 145 = ATCC 43992
30


Shewanella colwelliana
AY653177
LST-W = ATCC 39565.
73


Shewanella algae
AF005249
OK-1 = ATCC 51192
45


Shewanella frigidimarina
U85903
ACAM 591 = ATCC
38




700753



Shewanella gelidimarina
U85907
ACAM 456 = ATCC
39




700752



Shewanella woodyi
CP000961
MS32 = ATCC 51908
84


Shewanella amazonensis
AF005248
SB2B = ATCC 700329
44


Shewanella baltica
AJ000214
CCUG 39356 = DSM
55




9439



Shewanella oneidensis
AF005251
MR-1 = ATCC 700550.
46


Shewanella pealeana
AF011335
ANG-SQ1 = ATCC
52




700345



Shewanella violacea
D21225
strain. DSS12 = CIP
42




106290



Shewanella japonica
AF145921
ATCC BAA-316
72


Shewanella denitrificans
AJ311964
OS217 = DSM 15013
34


Shewanella
AJ300834
NF22 = LMG 19866.
40


livingstonensis





Shewanella olleyana
AF295592
ACEM 9 = LMG 21437
83


Shewanella fidelis
AF420312
ATCC BAA-318
36


Shewanella marinintestina
AB081757
IK-1 = JCM 11558
47


Shewanella sairae
AB081762
SM2-1 = JCM 11563.
49


Shewanella schlegeliana
AB081760
HRKA1 = JCM 11561
48


Shewanella waksmanii
AY170366
ATCC BAA-643
54


Shewanella affinis
AY351983
ATCC BAA-642
61


Shewanella aquimarina
AY485225
SW-120 = JCM 12193
67


Shewanella gaetbuli
AY190533
TF-27 = JCM 11814
56


Shewanella marisflavi
AY485224
SW-117 = JCM 12192
66


Shewanella pacifica
AF500075
R10SW1 = DSM 15445
59


Shewanella profunda
AY445591
LT13a = DSM 15900
64


Shewanella decolorationis
AJ609571
S12 = JCM 21555
65


Shewanella
AB204519
SCRC-2738 = JCM
79


pneumatophori

13187.



Shewanella sediminis
CP000821
HAW-EB3 = DSM 17055
89


Shewanella abyssi
AB201475
c941 = DSM 17171
74


Shewanella hafniensis
AB205566
P010 = ATCC BAA-1207
75


Shewanella halifaxensis
AY579751
HAW-EB4 = DSM 17350
70


Shewanella irciniae
DQ180743
UST040317-058 = JCM
81




13528



Shewanella kaireitica
AB094598
c931 = DSM 17170
58


Shewanella loihica
DQ286387
PV-4 = ATCC BAA-1088
82


Shewanella morhuae
AB205576
U1417 = ATCC BAA-1205
78


Shewanella spongiae
DQ167234
HJ039 = JCM 13830
80


Shewanella surugensis
AB094597
c959 = DSM 17177
57


Shewanella algidipiscicola
AB205570
S13 = LMG 23746
76


Shewanella atlantica
AY579752
HAW-EB5 = CCUG
71




54554



Shewanella canadensis
AY579749
HAW-EB2 = CCUG
68




54553



Shewanella donghaensis
AY326275
LT17 = JCM 12524
60


Shewanella
AB205571
T147 = LMG 23744
77


glacialipiscicola





Shewanella haliotis
EF178282
DW01 = JCM 14758.
33


Shewanella piezotolerans
AJ551090
WP3 = JCM 13877.
63


Shewanella psychrophila
AJ551089
WP2 = JCM 13876.
62


Shewanella basaltis
EU143361
J83 = JCM 14937
35


Shewanella chilikensis
FM210033
JC5 = CCUG 57101
51


Shewanella marina
EU290154
C4 = JCM 15074
37


Shewanella vesiculosa
AM980877
M7 = CECT 7339
41


Shewanella corallii
FJ041083
fav-2-10-05 = DSM 21332
53


Shewanella fodinae
FM203122
JC15 = CCUG 57102
50


Shewanella xiamenensis
FJ589031
S4 = JCM 16212.
43


Shewanella algae
X81621
BrY = ATCC 51181
27


Shewanella baltica
NC_009052
OS155 = ATCC BAA-
88




1091



Shewanella putrefaciens
NC_009438Error!
CN-32 = ATCC BAA-453
87



Bookmark not
or ATCC BA-1097




defined.
















TABLE 14





Sequences that are underlined and bold are unique signatures different from the Shewanella consensus.


Differences between MPHPW-1 and BrY are italicized for MPHPW-1. SEQ ID NOs are in parentheses.















































TABLE 15







Listing of sequences with highest sequence identity to


the 16S rDNA of MPHPW-1 identified in a BLAST search











Percent
Alignment
Identified


Accession (Seq ID)
Identity
Length
Organism





FJ866783(91)
99.79
1423
Shewanella algae strain





PSB-05


HQ851081(92)
99.79
1423
Rhodobacter





capsulatus** strain





NBY31


US20100044304
99.79
1423
Shewanella algae strain


SEQ ID 3 (26)


IBI-6P IPOD No. FERM





BP-10568


FJ866781(93)
99.72
1423
Shewanella algae strain





PSB-04


GU223381 (94)
99.72
1423
Shewanella sp EM0501


HM016084 (95)
99.72
1423
Shewanella sp KJW27


X81621(25)
99.72
1423
Shewanella algae BrY





**Apparently misclassified as Rhodobacter capsulatus






Example 27
Riboprint® Analysis of MPHPW-1
To determine whether the 16S rDNA genomic region of Shewanella algae strain MPHPW-1 contained additional distinguishing elements from Shewanella algae BrY, other Shewanella algae strains and a Shewanella chilikensis strain, several genomic DNA preparations of this strain and six other Shewanella algae or algae-like strains were analyzed by Riboprinter® These Riboprints® were compared to 7525 patterns contained within DuPont Environmental Services and Qualicon libraries compiled from samples taken from DuPont as well as another 6950 patterns that DuPont Qualicon has supplied from standard identified organisms. Based on the analyses of Riboprint® Batch 1074 (FIG. 25), which provides a chromosomal fingerprint of 16S rDNA loci of the tested strains, it is clear that the Riboprint® pattern for strain MPHPW-1 (Sample 1) is unique when compared against the six other strains assayed. The MPHPW-1 Riboprint® pattern was also unique among the available DuPont Riboprint® Libraries. The MPHPW-1 pattern was designated as RiboGroup® Identifier 212-1074-S-1. It is probable for various strains to share single similar Riboprint® bands generated by hybridizing the labeled E. coli rDNA operon probe to each strain's genomic Eco RI fragments, but it is the overall Riboprint® banding pattern that constitutes identification of a given strain in a specific Riboprint® or Ribogroup® identifier.
This analysis confirmed that the genomic sequences surrounding the rDNA operons in strain MPHPW-1 have different genomic structure than those in the Riboprint® database. Shewanella algae strain BrY, whose sequence identity to strain MPHPW-1 is 99.72% has a Riboprint® pattern that is similar to that of MPHPW-1, but its pattern is missing the 1 kb band (fragment). The region for this band is circled in FIG. 25 to demonstrate its absence in row 6, which contains the Riboprint® pattern for Shewanella algae strain BrY. Therefore strain MPHPW-1's Riboprint® is a unique genomic identifier, indicating that MPHPW-1 is a newly identified strain.

## Claims

1. A composition for enhanced oil recovery comprising a mixture of:
a) at least one Shewanella sp. that has been isolated and purified from its natural environment, wherein said Shewanella sp. comprises a 16S rDNA comprising SEQ ID NO: 24 and SEQ ID NO: 28; and
b) a synthetic growth medium that has been supplemented with electron acceptors selected from the group consisting of nitrate, fumarate, ferric ion, manganese (MnIV) ion and mixtures thereof.

2. The composition of claim 1 wherein the Shewanella sp. Further comprises a Riboprint® pattern identifier of 212-1074-S-1 as illustrated in FIG. 25.

3. The Shewanella sp. of claim 2 wherein the Shewanella sp. is MPHPW-1 (ATCC No. PTA-11920).

4. The composition of claim 1 further comprising at least one carbon source.

5. The composition of claim 1 further comprising at least one additional microorganism.