US 20120277127A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0277127 A1 Hendrickson et al. (43) Pub. Date: Nov. 1, 2012

(54) METHODS, STRAINS, AND COMPOSITIONS Publication Classification USEFUL FOR MICROBALLY ENHANCED Int. C. OIL RECOVERY ARCOBACTER CLADE 1 (51) C09K 8/582 (2006.01) (75) Inventors: Edwin R. Hendrickson, Hockessin, CI2N L/20 (2006.01) DE (US); Scott Christopher Jackson, Wilmington, DE (US); Abigail K. Luckring, (US) (52) U.S. Cl...... 507/201:435/252.1 (73) Assignee: E. DUPONT DE NEMOURS AND COMPANY, Wilmington, DE (US) (57) ABSTRACT Appl. No.: Methods, microorganisms, and compositions are provided (21) 13/280,972 wherein oil reservoirs are inoculated with microorganisms (22) Filed: Oct. 25, 2011 belonging to Arcobacter clade 1 and medium including an electron acceptor. The Arcobacter strains grow in the oil Related U.S. Application Data reservoir to form plugging biofilms that reduce permeability (60) Provisional application No. 61/408,739, filed on Nov. in areas of Subterranean formations thereby increasing Sweep 1, 2010. efficiency, and thereby enhancing oil recovery. Patent Application Publication Nov. 1, 2012 Sheet 1 of 12 US 2012/0277127 A1

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METHODS, STRAINS, AND COMPOSITIONS 0005 Additional useful microbial strains and methods for USEFUL FOR MICROBALLY ENHANCED enhancing oil recovery are needed to further improve the OIL RECOVERY ARCOBACTER CLADE 1 recovery of oil from oil reservoirs.

SUMMARY OF THE INVENTION 0001. This application claims the benefit of U.S. Provi 0006. The invention relates to methods for enhancing oil sional Application 61/408,739, filed Nov. 1, 2010 and is recovery from an oil reservoir, as well as to isolated micro incorporated by reference in its entirety. organisms and compositions that may be used to enhance oil recovery. 0007 Accordingly, the invention provides a method for FIELD OF THE INVENTION enhancing oil recovery from an oil reservoir comprising: 0008 a) providing a composition comprising: 0002. This disclosure relates to the field of environmental 0009 i) at least one strain of Arcobacter belonging to microbiology and modification of crude oil well properties Arcobacter clade 1; and using microorganisms. More specifically, methods for 0010 ii) a minimal growth medium comprising at improving oil recovery from an underground reservoir are least one electron acceptor, presented and new microorganisms are identified that can be 0.011 b) providing an oil reservoir; used for oil recovery. 0012 c) inoculating the oil reservoir with the composi tion of (a) Such that the Arcobacter containing compo BACKGROUND OF THE INVENTION sition populates and grows in the oil reservoir, and 0013 d) recovering oil from the oil reservoir; 0003. During recovery of oil from oil reservoirs, typically 0.014 wherein growth of the Arcobacter in the oil res only a minorportion of the original oil in the oil-bearing strata ervoir enhances oil recovery. is recovered by primary recovery methods which use only the 0015. In one embodiment, the strain of Arcobacter natural forces present in an oil reservoir. To improve oil belonging to Arcobacter clade 1 comprises the 16S rDNA recovery, a variety of Supplemental recovery techniques such degenerate consensus sequence of SEQID NO:40. as water flooding, which involves injection of water through 0016. In another embodiment, the strain of Arcobacter well bores into the oil reservoir, have been used. As water belonging to Arcobacter clade 1 comprises a 16S rDNA moves into the reservoir from an injection well and moves sequence having at least about 97% sequence identity to a through the reservoir strata, it displaces oil to one or more sequence selected from the group consisting of SEQID NOs: production wells where the oil is recovered. One problem 1, 2, 3, 4, 5, 6, 7, 8, and 39. commonly encountered with water flooding operations is 0017. In yet another embodiment, the invention provides poor sweep efficiency of injection water. Poor sweep effi an isolated microorganism of a strain selected from the group consisting of 97 AE3-3 (ATCC No. PTA-11410) and 97 AE3 ciency occurs when water preferentially channels through 12 (ATCC No. PTA-11409). highly permeable Zones of the oil reservoir as it travels from 0018. In yet another embodiment, the invention provides the injection well(s) to the production well(s), thus bypassing an oil recovery enhancing composition comprising: less permeable oil-bearing strata. Oil in the less permeable 0.019 a) at least one isolated strain of Arcobacter com Zones is thus not recovered. Poor Sweep efficiency may also prising a partial 16S rDNA sequence selected from the be due to differences in the mobility of the water versus that group consisting of SEQID NOs;1, 33, 34, 35, 36, 37, of the oil. and 38: 0004 Microorganisms have been used to enhance oil 0020 b) one or more electron acceptors; and recovery from Subterranean formations using various pro 0021 c) at least one carbon source. cesses which may improve Sweep efficiency and/or oil release. For example, viable microorganisms may be injected BRIEF DESCRIPTION OF FIGURES AND into an oil reservoir where they may grow and adhere to the SEQUENCES Surfaces of pores and channels in the rock or sand matrices in the permeable Zones to reduce water channeling, and thereby 0022. The invention can be more fully understood from targetinjection waterflow towards less permeable oil-bearing the following detailed description, the Figures, and the strata. Processes for promoting growth of indigenous accompanying sequence descriptions, which form a part of microbes by injecting nutrient solutions into Subterranean this application. formations are disclosed in U.S. Pat. No. 4,558,739 and U.S. 0023 FIG. 1 shows a molecular phylogenetic tree for Pat. No. 5,083.611. Injection of microorganisms isolated Arcobacter species and related bacteria based on 16S rRNA from oil recovery sites into Subterranean formations along gene sequences (rDNA), separating the described Arcobacter with nutrient solutions has been disclosed, including for spp. into at least three phylogenetic clades. Pseudomonas putida and Klebsiella pneumoniae (U.S. Pat. 0024 FIG. 2 shows a molecular phylogenetic tree for No. 4,800.959), for a Bacillus strain or Pseudomonas strain newly isolated Arcobacter species and reference bacteria I-2 (ATCC 30304) isolated from tap water (U.S. Pat. No. based on 16S rRNA gene sequences (rDNA). 4.558,739), and for Pseudomonas putida, Pseudomonas (0025 FIG. 3A-D shows an alignment of 16S rDNA aeruginosa, Corynebacterium lepus, Mycobacterium rhodo sequences for Arcobacter clade 1 dominant consensus, Arco chrous, and Mycobacterium vaccae (U.S. Pat. No. 5,163, bacter clade 1 degenerate consensus, strain 97AE3-12, Arco 510). Injection of isolated microorganisms and a surfactant is bacter clade 2 degenerate consensus, and Arcobacter clade 3 disclosed in U.S. Pat. No. 5,174,378. degenerate consensus. US 2012/0277127 A1 Nov. 1, 2012

0026 FIG. 4 shows a RiboprinterR) analysis of various Arcobactorsp Strains. TABLE 1-continued 0027 FIG. 5 shows dominate and degenerate signature sequences for Shewanella species in rDNA variable regions 2 16S rDNA seqs of Arcobacter strains including coordinates (A), 5 (B), and 8 (C). The variable positions are underlined. 8 to 1511 in the E. coli 16S rRNA sequence. Alternative nucleotides for each variable position designation SEQ are given in the legend. Species Strain Identification ID NO 0028 FIG. 6 shows a schematic diagram of the slim tube Arcobacter CCUG 10373T Type strain 21 experimental set up used to measure plugging of permeable butzierii sand packs Arcobacter RM4O18 isolate 22 butzierii 0029 FIG. 7 shows a graph of the pressure drop across a Arcobacter sp R-28214 isolate 23 non-inoculated slim tube. Arcobactersp. clone PL-7C7 clone 24 Arcobactersp. clone PL-8B1 clone 25 0030 FIG. 8 shows a graph of the pressure drop across a Arcobacter 1621.54 isolate 26 slim tube that was inoculated with Arcobacter sp97AE3-12 sulfidicus (ATCC NO: PTA-11409) and then batch fed periodically. Uncultured clone BP-B88 clone 27 bacterium 0031 FIG. 9 shows a graph of the pressure drop across a Oilfield FWKOB isolate 28 slim tube that was inoculated with Arcobacter sp97AE3-12 bacterium (ATCC NO: PTA-11409) and then continuously fed. Sulfurospirillum Strain K, Type strain 29 0032. The following sequences conform with 37 C.F.R. multivorans DSM 12446T Uncultured CIOle clone 30 SS 1.821-1.825 (“Requirements for Patent Applications Con bacterium ASO77 B63 taining Nucleotide Sequences and/or Amino Acid Sequence Thiomicrospirasp Strain CVO isolate 31 Disclosures—the Sequence Rules”) and are consistent with "An isolate is a colony isolated from a sample World Intellectual Property Organization (WIPO) Standard *A clone contains a PCR amplified fragment generated from bacterial DNA isolated from a ST.25 (2009) and the sequence listing requirements of the sample, which is sequenced to determine the make up of a population EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and 0033 SEQ ID NO:32 is a partial E. coli 16S rDNA format used for nucleotide and amino acid sequence data sequence used in alignments of Arcobacter 16S rDNA comply with the rules set forth in 37 C.F.R. S1.822. Sequences. 0034 SEQIDNO:33 is apartial 16S rDNA sequence from TABLE 1. Arcobacter sp. 97AE3-1. 0035) SEQID NO:34 is apartial 16S rDNA sequence from 16S rDNA seqs of Arcobacter strains including coordinates Arcobacter sp. 97 AE3-3. 8 to 1511 in the E. coli 16S rRNA sequence. 0036 SEQID NO:35 is apartial 16S rDNA sequence from SEQ Arcobacter sp. 97 AE3-7. Species Strain Identification ID NO 0037 SEQID NO:36 is apartial 16S rDNA sequence from Arcobacter sp strain Isolate" 1 Arcobacter sp. 97AE4-1. 97AE3-12 0038 SEQID NO:37 is apartial 16S rDNA sequence from Arcobactersp. Solar Lake isolate 2 isolate Arcobacter sp. 97AE4-5. Arcobactersp. clone YJO-18 clone 3 0039 SEQID NO:38 is apartial 16S rDNA sequence from Arcobacter CL-S1, Type strain 4 Arcobacter sp. 97AE4-6. marinus JCM 15502; Arcobactersp. clone EB27.1 clone 5 0040 SEQID NO:39 is a dominant consensus sequence Arcobacter LA31BT Type strain 6 for Arcobacter sp. clade 1 16S rDNA. halophilus 0041 SEQID NO:40 is a degenerate consensus sequence Arcobacter F98-3 T Type strain 7 for Arcobacter sp. clade 1 16S rDNA. molluscorum Arcobacter F2075T, Type strain 8 0042 SEQID NO:41 is a degenerate consensus sequence mytili for Arcobacter sp. clade 2 16S rDNA. Arcobacter CCUC 15893 Type strain 9 nitrofigilis 0043 SEQID NO:42 is a degenerate consensus sequence Arcobacter SW28-11T Type strain 10 for Arcobacter sp. clade 3 16S rDNA. defluvii 0044 SEQ ID NOs:43-46 are primers 1492R, 8F, M13 Arcobactersp. R-283141 isolate 11 Reverse, and M13 Forward, respectively. Arcobacter sp KTO913 isolate 12 Arcobacter sp BSs2O195 isolate 13 0045 SEQID NO:47 is the Shewanella dominant signa Arcobacter sp clone A3b2 clone 14 ture sequence for the 16S rDNA variable region 2. Arcobacter DSM 7299T Type strain 15 nitrofigilis 0046 SEQID NO:48 is the Shewanella degenerate signa Arcobacter F2176 isolate 16 ture sequence for the 16S rDNA variable region 2. nitrofigilis 0047 SEQID NO:49 is the Shewanella dominant signa Arcobacter CCUG 17082T Type strain 17 cryaerophilus ture sequence for the 16S rDNA variable region 5. Arcobacter LMG 21996T Type strain 18 0048 SEQID NO:50 is the Shewanella degenerate signa cibarius ture sequence for the 16S rDNA variable region 5. Arcobacter 6695-3 Type strain 19 thereius 0049 SEQID NO:51 is the Shewanella dominant signa Arcobacter CCUG 10374T Type strain 2O ture sequence for the 16S rDNA variable region 8. Skirrowi 0050 SEQID NO:52 is the Shewanella degenerate signa ture sequence for the 16S rDNA variable region 8. US 2012/0277127 A1 Nov. 1, 2012

0051) Applicants made the following biological deposits 0062. The abbreviation “DNA refers to deoxyribonucleic under the terms of the Budapest Treaty on the International acid. Recognition of the Deposit of Microorganisms for the Pur 0063. The abbreviation “ATCC refers to AmericanType poses of Patent Procedure: Culture Collection International Depository, Manassas, Va., USA. “ATCC No.” refers to the accession number to cultures TABLE 2 on deposit with ATCC. 0064. The abbreviation “CCUG' refers to the Culture Col Information on deposited strains lection of the University of Göteborg, Sweden, which is a Depositor Identification International Depository Date of collection of microorganisms. Reference Designation Deposit 0065. The abbreviation “DSM or “DSMZ refers to Arcobacter sp97AE3-3 ATCC No. PTA-11410 Oct. 14, 2010 Deutsche Sammlung Von Mikroorganismen and Zellkulturen Arcobacter sp97AE3-12 ATCC No. PTA-11409 Oct. 14, 2010 GmbH which is a German collection of microorganisms and cell cultures (Braunschweig, Germany). 0066. The terms “oil well, “oil reservoir, and “oil-bear ing stratum may be used herein interchangeably and refer to DETAILED DESCRIPTION OF THE INVENTION a subterranean or sub sea-bed formation from which oil may 0052 Applicants specifically incorporate the entire con be recovered. The formation is generally a body of rocks and tent of all cited references in this disclosure. Unless stated soil having Sufficient porosity and permeability to store and otherwise, all percentages, parts, ratios, etc., are by weight. transmit oil. Trademarks are shown in upper case. Further, when an 0067. The term “well bore refers to a channel from the amount, concentration, or other value or parameter is given as Surface to an oil-bearing stratum with enough size to allow for either a range, preferred range or a list of upper preferable the pumping of fluids either from the surface into the oil values and lower preferable values, this is to be understood as bearing stratum (injection well) or from the oil-bearing Stra specifically disclosing all ranges formed from any pair of any tum to the surface (production well). upper range limit or preferred value and any lower range limit 0068. The terms “denitrifying and “denitrification” mean or preferred value, regardless of whether ranges are sepa reducing nitrate for use in respiratory energy generation. rately disclosed. Where a range of numerical values is recited 0069. The term “sweep efficiency” refers to the fraction of herein, unless otherwise Stated, the range is intended to an oil-bearing stratum that has seen fluid or water passing include the endpoints thereof, and all integers and fractions through it to move oil to production wells. One problem that within the range. It is not intended that the scope of the can be encountered with waterflooding operations is the rela invention be limited to the specific values recited when defin tively poor sweep efficiency of the water, i.e., the water can ing a range. channel through certain portions of a reservoir as it travels 0053. The invention relates to methods for enhancing oil from injection well(s) to production well(s), thereby bypass recovery from an oil reservoir by inoculating an oil reservoir ing other portions of the reservoir. Poor sweep efficiency may with a strain of Arcobacter that by molecular phylogenetic be due, for example, to differences in the mobility of the water analysis of the 16S rDNA sequence belongs to Arcobacter versus that of the oil, and permeability variations within the clade 1 (defined herein), and a minimal growth medium that reservoir which encourage flow through some portions of the Supports growth of said Arcobacter under denitrifying con reservoir and not others. ditions in the subterranean location. Growth of said Arco (0070. The term “pure culture” means a culture derived bacter in the oil reservoir may form biofilms that plug more from a single cell isolate of a microbial species. The pure permeable Zones in sand or sandstone layers thereby rerout cultures specifically referred to herein include those that are ing water towards less permeable, more oil rich areas. Sweep publicly available in a depository, and those identified herein. efficiency is thereby enhanced, leading to increased oil recov (0071. The term “biofilm’ means a film or “biomass layer” ery. of microorganisms. Biofilms are often embedded in extracel 0054. In addition, the invention relates to previously lular polymers, which adhere to Surfaces Submerged in, or unknown microorganisms isolated from water samples Subjected to, aquatic environments. Biofilms consist of a obtained from an oil reservoir and compositions containing matrix of a compact mass of microorganisms with structural any of these microorganisms, or other Arcobacter of clade 1. heterogeneity, which may have genetic diversity, complex which are useful in oil recovery methods. Improving oil community interactions, and an extracellular matrix of poly recovery using the described methods and microorganisms meric Substances. would increase the output of active oil wells. 0072 The term “plugging biofilm’ means a biofilm that is 0055. The following definitions are provided for the spe able to alter the permeability of a porous material, and thus cial terms and abbreviations used in this application: retard the movement of a fluid through a porous material that 0056. The term "PCR" refers to Polymerase chain reac is associated with the biofilm. tion. (0073. The term “simple nitrates” and “simple nitrites” 0057 The term “dNTPs” refers to Deoxyribonucleotide refer to nitrate (NO) and nitrite (NO), respectively, as triphosphates. they occur in ionic salts such as potassium nitrate, Sodium nitrate, and Sodium nitrite. 0058. The term “ASTM refers to the American Society (0074 The term “injection water” refers to fluid injected for Testing and Materials. into oil reservoirs for secondary oil recovery. Injection water 0059. The abbreviation “NCBI' refers to the National may be supplied from any suitable source, and may include, Center for Biotechnology Information. for example, Sea water, brine, production water, water recov 0060. The abbreviation “BSL refers to Biosafety Level. ered from an underground aquifer, including those aquifers in 0061. The abbreviation “RNA refers to ribonucleic acid. contact with the oil, or Surface water from a stream, river, US 2012/0277127 A1 Nov. 1, 2012 pond or lake. As is known in the art, it may be necessary to homologies of 16S rDNAS, including of signature sequences remove particulate matter including dust, bits of rock or sand in the 16S rDNA, and shows relationships of the present and corrosion by-products Such as rust from the waterprior to strains to related Strains and species. injection into the one or more well bores. Methods to remove I0084. The term “phylogenetic clade' or “clade” refers to a Such particulate matter include filtration, sedimentation and branch in a phylogenetic tree. A clade includes all of the centrifugation. related organisms that are located on the branch, based on the chosen branch point. 0075. The term “production water” means water recov I0085. The term “genomovar is used to describe a sub ered from production fluids extracted from an oil reservoir. species classification which is used when a group of strains of The production fluids contain both water used in secondary a species are differentiable by DNA sequence, but are pheno oil recovery and crude oil produced from the oil reservoir. typically indistinguishable. Genomovars are defined and 0076. The term “inoculating an oil well' means injecting identified by DNA-DNA hybridization and/or by 16S rDNA one or more microorganisms or microbial populations or a signature sequences. This terminology has been used to consortium into an oil well or oil reservoir such that micro describe Pseudomonas stilted by Bennasar et al. (1996) Int. organisms are delivered to the well or reservoir without loss J. of Syst. Bacteriol. 46:200-205). of viability. I0086. The term “ribotyping means fingerprinting of 0077. The term “phylogenetic typing”, “phylogenetic genomic DNA restriction fragments that contain all or part of mapping, or “phylogenetic classification may be used inter the genes coding for the 16S and 23S ribosomal RNAs. changeably herein and refer to a form of classification in Ribotyping is performed using the DuPont RiboPrinterR sys which microorganisms are grouped according to their evolu tem. tionary genetic lineage. Phylogenetic typing herein is of I0087. The term “RiboPrintTM” refers to the unique strains of microorganisms isolated from environmental genomic fingerprint of a specific microbial isolate or strain, samples and is based on 16S ribosomal RNA (rRNA) encod generated using the DuPont RiboPrinter(R) system. ing gene (rDNA) sequences. I0088. The term “type strain” refers the reference strain for a particular species whose description is used to define and 0078. The term “hypervariable regions” as used herein characterize a particular species. refers to sequence regions in the 16S rRNA gene where the I0089. The term “sequence analysis software” refers to any nucleotide sequence is highly variable. In most microbes the computer algorithm or software program that is useful for the 16S rDNA sequence consists of nine hypervariable regions analysis of nucleotide or amino acid sequences. “Sequence that demonstrate considerable sequence diversity among dif analysis software may be commercially available or inde ferent bacterial genera and species and can be used for genus pendently developed. Typical sequence analysis software and species identification includes, but is not limited to: the GCG suite of programs 007.9 The term “signature sequences” as used herein (Wisconsin Package Version 9.0, Genetics Computer Group refers to specific nucleotides at specific 16S rRNA encoding (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX gene (rDNA) positions (signature positions), which usually (Altschulet al., J. Mol. Biol. 215,403-410, 1990), DNASTAR occur within the hyperVariable regions, that are distinguish (DNASTAR, Inc., Madison, Wis.), and the FASTA program ing for microorganisms at different levels. At the signature incorporating the Smith-Waterman algorithm (Pearson, W. positions, nucleotides that distinguish between species may R., Comput. Methods Genome Res., Proc. Int. Symp, Meeting be one or more specific base Substitutions, insertions or dele Date 1992, 111-120, Eds: Suhai, Sandor, Plenum Publishing, tions. When taken together, the signature sequences of 16S New York, N.Y., 1994). Within the context of this application, rDNA are useful for describing microbes at the species, strain it will be understood that, where sequence analysis software or isolate level and can be used in the identification of a is used for analysis, the results of the analysis will be based on microbe. the “default values' of the program referenced, unless other 0080. The term “degeneracy or degenerate base position' wise specified. As used herein “default values' will mean any refers to the case where more than one nucleotide (A, G, C, or set of values or parameters which originally load with the T(U)) is possible at a particular position in a DNA or RNA software when first initialized. sequence. A position is a “two-fold degenerate site if only 0090 The term “electron acceptor refers to a compound two of four possible nucleotides may be at that position. A that receives or accepts an electron(s) during cellular respi position is a “three-fold degenerate' site if three of four ration. Microorganisms obtain energy to grow by transferring possible nucleotides may be at that position. A position is a electrons from an “electron donor to an electron acceptor. “four-fold degenerate site if all four nucleotides may be at During this process, the electron acceptor is reduced and the that position. electron donor is oxidized. Examples of electron acceptors include oxygen, nitrate, fumarate, iron (III), manganese (IV), 0081. The term “degenerate signature sequence” refers to Sulfate and carbon dioxide. Sugars, low molecular weight a signature sequence that may have one or more possible organic acids, carbohydrates, fatty acids, hydrogen and crude degenerate base positions in the signature sequence. oil or its components such as petroleum hydrocarbons or I0082. The term “phylogenetics” refers to the field of biol polycyclic aromatic hydrocarbons are examples of com ogy that deals with identifying and understanding evolution ary relationships between organisms, and in particular pounds that can act as electron donors. molecular phylogenetics uses DNA sequence homologies in 0091 “Darcy” is a unit of permeability. A medium with a this analysis. In particular, similarities or differences in 16S permeability of 1 darcy permits a flow of 1 cm/s of a fluid rDNA sequences, including signature sequences, identified with viscosity 1 c (1 mPas) under a pressure gradient of 1 using similarity algorithms serves to define phylogenetic atm/cm acting across an area of 1 cm. A millidarcy (mD) is relationships. equal to 0.001 darcy. I0083. The term “phylogenetic tree” refers to a branched Isolated Microorganisms diagram depicting evolutionary relationships among organ 0092 Microorganisms capable of growth under anaerobic isms. The phylogenetic tree herein is based on DNA sequence conditions in the presence of nitrate, fumarate or the ferricion US 2012/0277127 A1 Nov. 1, 2012

(Fe (III)) as an electron acceptor and lactate as a carbon NOs: 1,33, 34, 35, 36, 37, and 38, respectively, Thus strains Source were isolated from production and injection waters of with the partial rDNA sequences of these new isolates (SEQ Well #2 that is located in the Wainwright field in the province ID NOS:1, 33, 34,35,36, 37, and 38) are Arcobacter clade 1 of Alberta, Canada. Well #2 has a salinity of about 65 parts per strains of the present method. thousand (ppt) in both production and injection waters, which (0097. The 16S rDNA sequences of strains in each of is about twice the salinity of seawater. clades 1, 2, and 3 were analyzed as groups to identify signa 0093 Isolated microorganisms were characterized by ture sequences at specified positions that may be used to analysis of their 16S ribosomal DNA (rDNA) sequences and distinguish the three clades. As described in Example 2 by fingerprinting of their genomic DNA restriction fragments herein, specific positions in the 16S rDNA sequence have that contain all or part of the genes coding for the 16S and 23S nucleotides that are characteristic for each Arcobacter clade, ribosomal RNAs (rRNAs; ribotyping). Isolated strains which may be fixed or may have some degeneracy, as listed in 97AE3-12,97AE 3-3, 97 AE3-197AE 4-6,97AE4-5,97AE Table 6. In addition, there may be an insertion or deletion at 3-7, and 97AE 4-1 were identified as new strains belonging to Some positions. The set (all positions together) of signature the genus Arcobacter. RiboPrintTM patterns for these strains sequences for each Arcobacter clade that are listed in Table 6 were distinct from the closely related strain Arcobacter halo differs from each of the other Arcobacter clades set of sig philus (ATCC BAA-1022), which has over 96% sequence nature sequences. The Arcobacter clade 1 16S rDNA domi identity with strain 97AE3-12 in the 16S rDNA (SEQ ID nant (most prevalent) consensus sequence (which may not be NOs:6 and 1, respectively). The RiboPrintTM patterns (FIG.4) full length) containing the signature sequences, is provided as indicate that the newly isolated Strains form 3 groupings: 1) SEQ ID NO:39. Known or newly isolated microbial strains 97AE3-12,97AE4-1, and 97 AE4-5; 2)97AE3-3 and 97 AE4 may be identified as belonging to Arcobacter clade 1, and thus 6; and 3) 97AE3-7 and 97 AE3-1. The strains 97AE3-12 and are strains of the present method, by having 16S rDNA with 97AE 3-3 were deposited herewith under the Budapest Treaty at least about 97%, 98%, or 99% sequence identity to SEQID as ATCC #PTA-11409 and ATCC #PTA-11410, respectively. NO:39. 0094 Further, the strains were characterized as belonging 0098. The degenerate signature sequences (including to Arcobacter clade 1 as determined by molecular phyloge insertion/deletion positions) are present in the degenerate netic analysis of 16S rDNA sequences described in Example consensus 16S rDNA sequence for clade 1 (SEQID NO:40), 2 herein. The phylogenetic tree produced by the analysis is the degenerate consensus 16S rDNA sequence for clade 2 shown in FIG. 1, with the newly isolated strains represented (SEQ ID NO:41), and the degenerate consensus 16S rDNA by strain 97AE3-12. The phylogenetic tree shows that three sequence for clade 3 (SEQ ID NO:42). Known or newly clades, or groupings, are formed of known Arcobacter spe isolated microbial strains may be identified as belonging to cies, which are boxed in the figure. Clade 3 includes patho Arcobacter clade 1, and thus are strains of the present method, genic strains such as Arcobacter butzleri and Arcobacter by having 16S rDNA that is of SEQ ID NO:40. In addition, cryaerophilus, which are classified as BSL2. Clade 2 includes known or newly isolated microbial strains may be identified Arcobacter nitrofigilis. Clade 1 includes the known species as belonging to Arcobacter clade 1, and thus are strains of the Arcobacter marinas, Arcobacter halophilus, Arcobacter mol present method, by having 16S rDNA that includes the clade luscorum and Arcobacter mytili. Any strain of Arcobacter 1 degenerate signature sequences listed for specific positions that belongs to clade 1 may be used in the present method. in Table 6. Clade 1 includes the strains listed above, as well as any strains (0099. The Arcobacter sp strains 97AE3-12, 97AE 3-3, that belong to the same clade that these strains belong to when 97AE3-1, 97AE 4-6, 97AE 4-5, 97AE 3-7, and 97AE 4-1 analyzed by molecular phylogenetics using 16S rDNA were found as shown in examples herein to have properties sequences as described herein. indicating their ability to enhance oil recovery by growing to 0.095 Strains of Arcobacter clade 1, which are strains of form plugging biofilms. The strains were able to form plug the present method, may be defined further as strains with ging biofilms in high salinity conditions (75 ppt). Strain rDNA sequences having at least about 97%, 98%, or 99% 97AE3-12 grew in the presence of petroleum oil, in both low sequence identity to the 16S rDNA sequences of any of those (15 ppt) and high salinity (64 ppt) denitrifying conditions. strains shown in clade 1 in FIG. 1: Arcobacter sp Solar lake Plugging biofilms were produced in low (15 ppt) and high (35 (SEQ ID NO:2), Arcobacter sp YJO-18 (uncultured clone: ppt and 68 ppt) Salinity media. Plugging biofilms were SEQID NO:3), Arcobacter marinus (SEQID NO:4), H temp formed with either batch or continuous nutrient feeding. In Oil reservoir clone EB27 (SEQ ID NO:5), Arcobacter halo addition, silica particle aggregation was demonstrated in high philus (SEQ ID NO:6), and Arcobacter mytili (SEQ ID salinity (64 ppt) media. NO:8). In addition, strains of the present method include 0100. These properties of the isolated Arcobacter clade 1 those with rDNA sequences having at least about 97%, 98%, strains demonstrate their use for forming biofilms to plug or 99% sequence identity to the 16S rDNA sequence of Arco highly permeable Zones in permeable sand or rock of oil bacter molluscorum F98-3T (SEQ ID NO:7), which also reservoirs. Plugging of hyperpermeable Zones may reroute belongs to clade 1. The 16S rRNA sequences of strains from water towards less permeable, more oil rich areas thereby different clades in FIG. 1 have sequence identities of less than enhancing Sweep efficiency leading to increased oil recovery. 96%. Oil Recovery Enhancing Compositions 0096. A molecular phylogenetic tree prepared herein shows the relatedness of the newly isolated strains 97AE3-12, 0101. The newly isolated Arcobacter strains 97AE3-12 97AE 3-1, 97AE3-3, 97AE 3-7, 97AE 4-1, 97AE 4-5, and (ATCC iPTA-11409), 97AE 3-3 (ATCC #PTA-11410), 97AE 4-6 to each other and to other clade 1 strains, as well as 97AE 4-6, 97AE 4-5, 97AE 3-7, and 97AE 4-1 described to one strain each of clades 2 and 3, in FIG. 2. The strains are above may be included as components in oil recovery enhanc closely related to each other in one branch of clade 1. These ing compositions which are an embodiment of the present strains are characterized by their rNA sequences: SEQ ID invention. Thus the present compositions include at least one US 2012/0277127 A1 Nov. 1, 2012

strain of Arcobacter that has a partial rDNA sequence of SEQ 0107 The dominant signature sequences in FIG. 5 are ID NO:1, 33, 34, 35, 36, 37, or 38, which are the rDNA those with the variable positions designated as the most fre sequences of these new strains. Each of the strains may be in quently found nucleotides in Shewanella species. Shewanella separate oil recovery enhancing compositions, or any combi are gram negative, gamma-proteobacteria, which have the nation of more than one of the strains may be in the same ability to reduce metals and are capable of additionally reduc composition. ing a wide range of terminal electronacceptors. These micro organisms gain energy to Support anaerobic growth by cou 0102. In addition to one or more of these new Arcobacter pling the oxidation of H or organic matter to the reduction of clade 1 strains, the present oil recovery enhancing composi a variety of multivalent metals, which leads to the precipita tion includes one or more electron acceptors and at least one tion, transformation, or dissolution of minerals. carbon source. In one embodiment the electron acceptor is 0108. The ability of Shewanella species to alter the wet nitrate. Nitrate is reduced to nitrite and/or to nitrogen during tability of a hydrocarbon coated surface leading to improved growth of the described Arcobacter strains. Nitrite may also oil recovery is disclosed in commonly owned and co-pending serve as an electron acceptor in the composition. In various US Patent Application Publication #2011/0030956, which is embodiments the electron acceptor is one or more ionic salts hereinincorporated by reference. In one embodiment an addi of nitrate, one or more ionic salts of nitrite, or any combina tional microorganism is Shewanella putrefaciens, tion of ionic salts of nitrate and nitrite. Shewanella sp. LH4:18 (ATCC No. PTA-8822; described in 0103) The carbon source may be a simple or a complex commonly owned U.S. Pat. No. 7,776,795), or Shewanella sp carbon-containing compound. The carbon Source may be L3:3 (ATCC No. PTA-10980; described in commonly owned complex organic matter Such as peptone, corn steep liquor, or and co-pending US Patent Application Publication No. 2011/ yeast extract. In another embodiment the carbon Source is a 0030956). simple compound such as citrate, fumarate, maleate, pyru 0109. In one embodiment Thauera sp. AL9:8 (ATCC Vate, Succinate, acetate, formate or lactate. iPTA-9497) is included in the present composition. Thauera 0104 Oil recovery enhancing compositions may include sp. AL9:8 was isolated from subsurface soil samples and was additional components which promote growth of and/or bio shown to be capable of growth under denitrifying conditions film formation by the microbial strains of the composition. using oil or oil components as the Sole source of carbon. This These components may include, for example, Vitamins, trace microorganism also has oil releasing activity (U.S. Pat. No. metals, salts, nitrogen, phosphorus, magnesium, buffering 7,708,065). chemicals, and/or yeast extract 0105. In one embodiment the oil recovery enhancing com Methods of Enhancing Oil Recovery positions include one or more additional microorganisms 0110. The present oil recovery enhancing compositions which grow in the presence of oil. The microorganisms may may be used to inoculate an oil reservoir leading to enhance use a component of oil as a carbon source, or when using an ment in oil recovery. In addition, compositions including at alternate carbon source their growth is not inhibited by the least one strain belonging to Arcobacter clade 1, as described presence of oil. Particularly useful are other microorganisms above, and a minimal growth medium including at least one that have properties which enhance oil recovery, Such as electron acceptor may be used to inoculate an oil reservoir to microorganisms that form biofilms or that release oil from enhance oil recovery. Typically one or more ionic salts of Surfaces. In one embodiment an additional microorganism in nitrate and/or nitrite are used as the electron acceptor. The the present composition is a microorganism of a Shewanella microorganisms of Arcobacter clade 1 in the composition species. Shewanella is a bacterial genus that has been estab include viable cells that populate and grow in the oil reservoir. lished, in part through phylogenetic classification by rDNA 0111. A minimal growth medium includes at least one and is fully described in the literature (see for example Fre carbon Source, and may include other components such as drickson et al., Towards Environmental Systems Biology Of Vitamins, trace metals, salts, nitrogen, phosphorus, magne Shewanella, Nature Reviews Microbiology (2008), 6(8), sium, calcium, and buffering chemicals. The carbon Source 592-603; Hau at al., Ecology And Biotechnology. Of The may be a simple or a complex carbon-containing compound, Genus Shewanella, Annual Review of Microbiology (2007), for example, 1) oil or an oil component, 2) complex organic 61,237-258). matter Such as peptone, corn steep liquor, or yeast extract; or 0106 There is at least about 89% sequence identity of 16S 3) simple compounds Such as citrate, fumarate, maleate, rDNA sequences among Shewanella species. Shewanella pyruvate, Succinate, acetate, formate or lactate. species have 16S rDNA which has the signature sequences of 0112 Any strain belonging to Arcobacter clade 1, as hypervariable regions 2 (SEQ ID NOs:47 and 48 are domi described above, may be used which forms plugging biofilms nant and degenerate sequences, respectively), (SEQID NOs: under anaerobic denitrifying conditions in the presence of 49 and 50 are dominant and degenerate sequences, respec petroleum oil. Strains of microorganisms that belong to Arco tively) and 8 (SEQ ID NOs: 51 and 52 are dominant and bacter clade 1, as described above, that may be used in the degenerate sequences, respectively) as shown in FIG. 5. The present methods may be identified by their 16S rDNA combination of the degenerate signature sequences for each sequences, which have the signature sequences described region defines Shewanella species, including some position above and listed in Table 6. In addition, Strains belonging to variations as shown in FIG. 5. Thus Shewanella sp. useful in Arcobacter clade 1, as described above, useful in the present the present invention are those that comprise within the 16s methods may be identified by one skilled in the art using rDNA the degenerate signature sequences as set forth in SEQ biofilm formation, silica aggregation, and/or permeability ID NOs:48, 50, and 52. In one embodiment Shewanella sp. reduction assays such as those described in Examples herein. useful in the present invention are those that comprise within As examples of strains belonging to Arcobacter clade 1, as the 16s rDNA the dominant signature sequences as set forth in described above, that are able to form plugging biofilms, SEQID NOs:47, 49, and 51. these properties of strains 97 AE3-12 (ATCC #PTA-11409), US 2012/0277127 A1 Nov. 1, 2012

97AE 3-3 (ATCC #PTA-11410), 97AE3-1,97AE 4-6,97AE have a Substantially higher permeability compared to the rest 4-5,97AE 3-7, and 97AE 4-1 are demonstrated herein. In one of the rock layers. The higher permeability layers will chan embodiment, any of these strains are used in the present nel water and prevent water penetration to the other parts of methods. the oil-bearing stratum. Formation of plugging biofilms by 0113. In another embodiment, one or more microorgan microorganisms will reduce this channeling. isms in addition to strains belonging to Arcobacter clade 1, as described above, which grow in the presence of oil under EXAMPLES denitrifying conditions, are included in a composition used in the present method. Microorganisms of Shewanella species, 0118. The present invention is further defined in the fol which are described above, are particularly useful. lowing Examples. It should be understood that these 0114. In certain oil reservoirs having specific properties, Examples, while indicating preferred embodiments of the specific strains belonging to Arcobacter clade 1, as described invention, are given by way of illustration only. From the above, may be best suited for use in the present methods. For above discussion and these Examples, one skilled in the art example, in oil reservoirs where at least one fluid, Such as can ascertain the essential characteristics of this invention, injection water and/or production water, has a high concen and without departing from the spirit and scope thereof, can tration of salt, strains belonging to Arcobacter clade 1, as make various changes and modifications of the invention to described above, which grow and form plugging biofilms in adapt it to various usages and conditions. high Salt media are particularly suitable. Specifically, Arco bacter clade 1 strains 97 AE3-12 (ATCC#PTA-11409),97AE General Methods 3-3 (ATCC #PTA-11410), 97AE3-1, 97AE 4-6, 97AE 4-5, 0119 The meaning of abbreviations are used in this appli 97AE 3-7, and 97AE 4-1 are particularly suited to oil reser cation are as follows: “hr” means hour(s), “min' means min Voirs with at least one fluid having high salt, particularly salt ute(s), “day’ means day(s), “mL or “ml” means milliliters, of about 30 ppt or higher. The salt concentration may be at “mg/mL means milligram per milliliter, “L” means liters, least about 30 ppt. 35 ppt. 40 ppt. 45 ppt. 50 ppt, 55 ppt 60 ppt. & G uL'99 means microliters, “mM”& G 99 means millimolar, “LL& G 99 65 ppt. 70 ppt, or 75 ppt, or higher. means micromolar, “nM means nano molar, “ug/L' means 0115 Oil reservoirs may be inoculated with compositions microgram per liter, “pmol” means picomol(s), “C.” means including one or more strain belonging to Arcobacter clade 1. degrees Centigrade, “F” means degrees Fahrenheit, “bp' as described above, and a minimal growth medium using any means base pair, “bps' means base pairs, “mm” means mil introduction method known to one skilled in the art. Typically limeter, ppm’ means part per million, g/L means gram per inoculation is by injecting a composition into an oil reservoir. liter, “mL/min' means milliliter per minute, “mL/hr” means Injection methods are common and well known in the art and milliliter per hour, “cfu/mL means colony forming units per any suitable method may be used (see for example Nontech milliliter, ''g' means gram, 'mg/L' means milligram per liter, nical guide to petroleum geology, exploration, drilling, and “Key means kilo or thousands of electron volts, “psi' means production, 2" edition. N.J. Hyne, Penn Well Corp. Tulsa, pounds (of force) per square inch, “LB' means Luria broth, Okla., USA, Freethey, G. W., Naftz, D. L., Rowland, R. C., & Davis, J. A. (2002); Deep aquifer remediation tools: Theory, “rpm” means revolution per minute, “NIC” means non-in design, and performance modeling, In: D. L. Naftz, S. J. oculated control. Morrison, J. A. Davis, & C. C. Fuller (Eds.); and Handbook of Growth of Microorganisms groundwater remediation using permeable reactive barriers (pp. 133-161), Amsterdam: Academic Press). Injection is I0120 Techniques for growth and maintenance of anaero typically through one or more injection wells, which are in bic cultures are described in “Isolation of Biotechnological communication underground with one or more production Organisms from Nature”. (Labeda, D. P. ed. 117-140, wells from which oil is recovered: McGraw-Hill Publishers, 1990). Nitrate, the ferric iron and fumarate are each individually utilized as the primary elec Enhanced Oil Recovery From An Oil Reservoir tronacceptor under the growth conditions used herein. Under denitrification, anaerobic growth is measured by nitrate 0116 Enhanced oil recovery in this context may include depletion from the growth medium over time. The reduction secondary or tertiary oil recovery of hydrocarbons from sub of nitrate to nitrogen has been previously described (Moreno surface formations. Specifically, hydrocarbons are recovered Vivian, C., et al., J. Bacterial., 181, 6573-6584, 1999). In that are not readily recovered from a production well by water Some cases nitrate reduction processes lead to nitrite accu flooding or other traditional secondary oil recovery tech mulation which is Subsequently further reduced to nitrogen. niques. Hence, accumulation and sometimes dissipation of nitrite is 0117 Primary oil recovery methods, which use only the therefore also considered evidence for active growth and natural forces present in an oil reservoir, typically obtain only metabolism by microorganisms. a minor portion of the original oil in the oil-bearing strata of an oil reservoir. Secondary oil recovery methods such as water flooding may be improved using methods herein which Determination of Viable Cell Titer (Most Probable Number) provide microorganisms and growth media for formation of I0121. In order to determine viable cell titer, samples from plugging biofilms in areas of Subterranean formations where cultures or slim tubes were diluted by 1:10 serial dilution in 8 there is a high variation in permeability. Biofilm plugging of rows per sample of a 96 well plate using standard Miller's the highly permeable regions of a reservoirreroute water used Luria Broth or Luria broth with 3.5% NaCl added. Titration in water flooding towards less permeable, more oil rich areas. was done using an automated Biomek200 robotic pipettor. Thus enhanced oil recovery is obtained particularly from oil Growth was determined by visual turbidity and recorded for reservoirs where sweep efficiency is low due to, for example, each of 8 rows. The most probable number algorithm of interspersion in the oil-bearing stratum of rock layers that Cochran (Biometrics (1950) pp 105-116) was used to deter US 2012/0277127 A1 Nov. 1, 2012

mine the viable cells/mL and the 95% confidence limits for rated dNTPs were removed using Edge Biosystems (Gaith this number in the original sample. ersburg, Md.) clean-up plates. Amplified reactions were 0122) The serial dilution method plating is used to deter pipetted into one well of a pre-spun 96 well clean up plate. mine the bacterial titer of such cultures. A series of 1:10 The plate was centrifuged for 5.0 min at 5,000xg in a Sorvall dilutions of Such samples is plated and the resulting colonies RT-7 (Sorvall, Newtown, Conn.) at 25°C. The cleaned up are counted. The number of colonies on a plate is then mul reactions were placed directly onto an Applied Biosystems tiplied by the dilution factor (the number of times that the 1:10 3730 DNA sequencer and sequenced with automatic base dilution was done) for that plate to obtain the bacterial count calling. in the original sample. I0129. Each of the assembled rDNA sequences was com pared to the NCBI rDNA database (about 260,000 rDNA Ion Chromatography sequences) using the BLAST algorithm (Altschulet al., Jour 0123 To quantitate nitrate and nitrite ions in aqueous nal of Molecular Biology, 1990). The highest scoring media, Applicants used an ICS2000 chromatography unit sequence identity hit was used as an identifier of the most (Dionex, Banockburn, Ill.). Ion exchange was accomplished closely related known species for Strain identification. on an AS15 anion exchange column using a gradient of 2 to 50 0.130. Alternatively, to generate amplified rDNA frag mM potassium hydroxide. Standard curves using known ments from individual strains, we chose primer sets from amounts of sodium nitrite or Sodium nitrate solutions were Grabowski et al. (FEMS Microbiology Ecology, 54:427–443 generated and used for calibrating nitrate and nitrite concen (2005)). The combination of primer SEQ ID NO:43 and trations. primer SEQ ID NO:44 was chosen to specifically amplify bacterial rDNA sequences. Measurement of Total Dissolved Salts by Refractometer I0131 The PCR amplification mix included: 1.0x GoTaq PCR buffer (Promega), 0.25 mM dNTPs, 25 pmol of each 0.124. The total dissolved salt was measured using a hand primer, in a 50 LL reaction volume. 0.5 LL of GoTaq poly held refractometer (Model RHS 10ATC, Huake Instrument merase (Promega) and 1.0LL (20 ng) of sample DNA were Co., Ltd). added. PCR reaction thermocycling protocol was 5.0 min at Samples from Oil Reservoir Production and Injection Waters 95° C. followed by 30 cycles of: 1.5 min at 95°C., 1.5 minat 0.125. A petroleum well system was sampled for this study 53°C., 2.5 min at 72° C. and final extension for 8 min at 72° that is called Well #2 in the Wainwright field in the province C. in a Perkin Elmer 9600 thermocycler (Waltham, Mass.). of Alberta, Canada. This well has a salinity of about twice The 1400 base pair amplification products were visualized on seawater, which is in the range of 65 ppt. Water samples were 1.0% agarose gels. The PCR reaction mix was used directly obtained from production and injection well heads as mixed for cloning into pCR-TOPO4 vector using the TOPO TA oil/water liquids in glass 1.0L brown bottles, filled to the top, cloning system (Invitrogen) as recommended by the manu capped and sealed with tape to prevent gas leakage. Gas from facturer. DNA was transformed into TOP10 chemically com inherent anaerobic processes Sufficed to maintain anaerobic petent cells selecting for amplicillin resistance. Individual conditions during shipment. The bottles were shipped in large colonies) were selected and grown in microtiter plates for plastic coolers filled with ice blocks to the testing facilities sequence analysis. Sequencing of the amplified fragments within 48 hr of sampling. and strain identification was as described above. DNA Preparation for Sequence Analysis Automated Ribotypinq 0126 Genomic DNA from bacterial colonies was isolated I0132 Automated ribotyping was used for conclusive by diluting bacterial colonies in 50 uL of water or Tris-HCL identification of selected strains with similar 16S rRNA buffer pH7-8. Diluted colony DNAs were amplified with Phi sequence phylogenetic characteristics (Webster, John A 29 DNA polymerase prior to sequencing (GenomiPHI (1988) U.S. Pat. No. 4,717,653; Bruce, J. L. (1996) Food Amplification Kit GE Life Sciences, New Brunswick, N.J.). Techno. 50: 77-81; and Sethi, M. R. (1997) Am. Lab. 5: An aliquot (1.0LL) of a diluted colony was added to 9.0LL of 31-35). Ribotyping was performed as recommended by the the Lysis Reagent (from the GenomiPHI Amplification Kit) manufacturer (DuPont Qualicon Inc., Wilmington, Del.). For and heated to 95°C. for 3 min followed by immediate cooling these analyses, one fresh colony was picked, resuspended in to 4°C.9.0 uL of Enzyme Buffer and 1.0L of Phi 29 enzyme the sample buffer and added to the processing module for the were added to each lysed sample followed by incubation at heat treatment step at 80°C. for 10 minto inhibit endogenous 30°C. for 18 hr. The polymerase was inactivated by heating to DNA-degrading enzymes. The temperature was then 65° C. for 10 min followed by cooling to 4°C. reduced, and two lytic enzymes (lysostaphin and N-acetyl muramidase; provided by the manufacturer) were added to DNA Sequence Analyses the sample. The sample carrier was then loaded onto the 0127 DNA sequencing reactions were set up as follows: RiboprinterTM system with the other commercial reagents. 8.0 pt of GenomiPHI amplified sample were added to 8.0 LL Restriction enzyme digestion of the sample chromosomal of BigDye v3.1 Sequencing reagent (Applied BioSystems, DNA using EcoRI enzyme, gel electrophoresis and blotting Foster City, Calif.) followed by 3.0 uL of 10 uM primers SEQ steps were completely automated. Briefly, genomic bacterial IDNOs:43, 44, 45, or 46 (prepared by Sigma Genosys, Wood DNA was digested with the EcoRI restriction enzyme and lands, Tex.), 4.0 uL of 5x BigDye Dilution buffer (Applied loaded onto an agarose gel. Restriction fragments were sepa Biosystems) and 17 uL Molecular Biology Grade water (Me rated by electrophoresis and simultaneously transferred to a diatech, Inc., Herndon, Va.). nylon membrane. After a denaturation step, the nucleic acids 0128 Sequencing reactions were heated for 3.0 min at 96° were hybridized with a sulfonated DNA probe harboring the C. followed by 200 thermocycles of (95°C. for 30 sec; 55° C. rRNA operon of E. coli, which includes genes for the small for 20 sec; 60° C. for 2 min) and stored at 4°C. Unincorpo and large rRNA subunits, the 5S rRNA gene, and the internal US 2012/0277127 A1 Nov. 1, 2012

transcribed spacers. The hybridized probe was detected by 0.138 Packing two identical slim tubes with a mixture capturing light emission from a chemiluminescent Substrate of sand produced from an oil well plus Sil-co-Sil 125 as with a charge-coupled device camera. The output consisted of described below a densitometric fingerprint scan depicting the distribution of 0.139 Flooding each slim tube, under pressure the genomic EcoRI restriction fragments containing 0140. Determining base permeability of packed slim sequences from the ribosomal operon(s) in the genome, that tubes by flowing brine (Brine #1) into the tubes. are electrophoretically separated by their molecular weights. Successful bioplugging of these slim tube apparatuses using Screening of Strains for their Ability to Form Biofilms on an Arcobacter inoculum Suggests its utility in modifying the Sintered Glass Filters permeability of porous rock of an oil reservoir. Application of 0133. An assay to screen for strains that could form bio this strain to oil reservoirs could therefore improve oil recov films on silica surfaces and prevent water flow through about ery by altering the flow conformance of reservoirs under 10 micron pore spaces (plugging) was developed using sin water flooding. tered glass filters. 25 mm medium coarseness sintered glass 0.141. A schematic diagram of the slim tube experimental filters (stock #15254, Adams and Chittenden Scientific Glass, set up is shown in FIG. 6. All numbers below in bold refer to Berkeley Calif.) were glued into the base of plastic holders FIG. 6. designed for membrane filtration. After curing, the filter 0142. A sample of sand that was produced from the assemblies were sterilized by autoclaving. Individual filters Schrader Bluff formation at the Milne Point Unit of the in holders were placed in sterile Petri plates and media which Alaska North Slope was cleaned by washing with a solvent contained inoculum from overnight cultures of various strains made up of a 50/50 (volume/volume) mixture of methanol was added on top of the glass filters. Growth medium for this and toluene. The solvent was Subsequently drained and then biofilm formation/plugging assay was as indicated in the evaporated off the sand to produce clean, dry, flowable sand. Examples. The plates were covered and incubated at room This sand was sieved to remove particles less than one temperature under anaerobic conditions for one to two weeks. micrometer in size. This sand was combined with washed The filters were removed from the culture medium and the top Sil-co-Sil 125 (U.S. Silica, Berkeley Springs, W.Va.) in a 4:1 piece screwed in place. A 1 mL Syringe attached to the inlet ratio, and the mixture was packed tightly into separate four port of the filter holder was filled with 1.0 mL of water and the foot (121.92 cm) long, about 1 cm inner diameter, flexible time to drain the water in the tube was measured in seconds. slim tubes (9a,9b) and compacted by vibration using a labo 0134. The sintered glass filters were prescreened for flow ratory engraver. rate before incubation with culture and the percent change in 0.143 Both ends of each slim tube were capped with com flow rate post incubation was determined at the end of the mon compression type fittings to keep the sand mix in it. experiment. Flexible /8 inch (0.32 cm) tubing capable of sustaining the pressures used in the test was attached to the fittings. The slim Screening of Strains for Aggregation of Silica tubes were mounted into a pressure vessel, (10) with the 0135 Arcobacter sp. strains were tested for their ability to tubing passing through the ends of the pressure vessel (11 and aggregate grains of crystalline silica. Crystalline silica repre 12) using commonly available pressure fittings (/8 inch (0.32 sents a Surrogate for the sand grains common to many Sub cm) union bulkhead) (18a, 18b and 21a, 21b). Additional terranean geological formations. A 100LL aliquot of crystal fittings and tubing were used to connect the inlet of each slim line silica 220 g/L (grain size range approximately 2-20 tube to a pressure pump (13a, 13b) and feed reservoir (14a, microns; Sil-co-Sil 125 made by U.S. Silica, Berkeley 14b). Other common compression fittings, including elbows Springs, W.Va.) was added to each sample tube. In addition, unions and tees, and tubing connected the inlet of each slim 8 mL of medium was added and the tubes were capped to tube to a transducer that measured the pressure above atmo restrict oxygen entry to the medium. spheric pressure (absolute pressure gauge) (20a, 20b). The 0136. Duplicate, live (inoculated) test treatments received inlet of the slim tube was also connected using the same types 200 uL of frozen stock of various strains as an inoculum. of tubing and fittings to the high pressure side of a commonly Also, uninoculated control tubes were set up that contained available differential pressure transducer (19a, 19b). Fittings all components, except the microbial inoculum. Tubes were and tubing connected the outlet of each slim tube to the low statically incubated at 30° C. Treatment tubes were mixed pressure side of the differential pressure transducer (19a, vigorously by 10 seconds of Vortexing. Turbidity increased 19b) and to a back pressure regulator (16a, 16b). The signals dramatically due to resuspension of the crystalline silica, from the differential pressure and the absolute pressure trans which had settled to the tube bottoms during incubation. The ducers were ported to a computer and these pressure readings decline in turbidity due to settling of the crystalline silica was were monitored and periodically recorded. The pressure ves monitored over time after mixing by measuring OD600. The sel (10) around the slim tubes was filled with water, which settling behavior of the silica particles showed that some acted as a hydraulic fluid, through a water port (15). This strains could form a strong adhesive interaction with adjacent water was slowly pressurized with air through port 17 to a crystalline silica particles, causing them to settle more rap pressure of about 107 pounds per square inch (psi) (0.74 mega Pascal) while Brine #1 (below) from the feed reservoirs (14a, idly. In the oil field, making sand grains adherent to one 14b) flowed through the slim tubes and came out through the another increases resistance to liquid flow through sand. This back pressure regulator (16a, 16b). This operation was per allows control over flow conformance which leads to more formed such that the pressure in each slim tube was always 5 efficient oil recovery via water flooding. to 20 psi (0.034-0.137 mega Pascal) below the pressure in the Slim Tube Apparatus for Permeability Reduction Assay pressure vessel (10). 0137 An apparatus was designed for measuring bioplug Solutions for Slim Tube Experiments: ging of permeable sand packs using slim tubes. The overall 0144 Brine #1: Injection water used at a well site in procedure for operating the slim tube was: Alberta Canada. The total dissolve salt content was about 70 US 2012/0277127 A1 Nov. 1, 2012

ppt. The pH of this solution was adjusted to about 6.2 to 6.4 AP=The pressure drop across a porous pack or rock, psi using HCl or NaOH. This brine was applied to slim tubes both Q=Volumetric flow rate through pack, =/hr with and without filter sterilization during the course of slim L=Viscosity of fluid (single phase) through pack = centi tube experiments as noted in the examples. po1se L-Length of pack (parallel to flow). = cm A=Cross sectional area (normal to flow) = cm Recipe 200 mL. k=Permeability = milliDarcy 4.08—a conversion constant to make the units compatible = Brine #2: (batch nutrient feed) mD-hr-psi/cp/cc NaNO 14.2 g 0149 Base permeability, along with other properties of NaLactate 7.2 g NHCI 720 mg each packed slim tube are given in Table 3. KH2PO 144 mg Yeast Extract 3.6 g. TABLE 3 pH = 6.5 Diluted 1 part in 36 of Brine #1 Properties of Sand packed slim tubes Brine #3: (continuous nutrient feed) perme NaNO, 14.2 g Tube Example Tube Length, Mass of ability, NaLactate 7.2 g i Number ID, cm L., cm Sand, g Darcy NHCI 720 mg KH2PO 144 mg 9a 8, 9 O.978 121.9 166.4 0.7 Yeast Extract 3.6 g. 9b 10 O.978 121.9 175.6 1.2 pH = 6.5 Diluted 1 part in 327 of Brine #1. Brine #5: Example 1 in Tap water, 66 ppt NaCl, 1000 ppm NaCitrate, Anaerobic Enrichment for Indigenous Microbes 3725 ppm Na Fumarate, from Oil Reservoir Samples 100 mg/L NHCl, 50 mg/L KH2PO4, 500 mg/L yeast extract 0150. To enrich for species that could reduce any of the 1900 ppm Ca (5260 ppm CaCl2) electron acceptors nitrate, fumarate or the ferric ion (Fe(III), Adjust the pH to ~6.2 to 6.4 with HCl. we inoculated 1 mL of either injection water or production water from Well #2, described in General Methods, into 9 mL of minimal salts media (Table 4) in 20 mL anaerobic serum vials, supplemented with lactate (2000 ppm) as the carbon Measurement of Pressure Drop Source, sodium chloride to 4300 ppm and as electronacceptor 0145 The pressure drop in the slim tubes was measured either 1.6 g/L Sodium nitrate, or 3.5 g/L Sodium fumarate, or using the differential pressure transducer described above. 13,000 ppm NaEDTAFe(III). A fourth enrichment having a The pressure drop was measured across each slim tube at rich medium, Marine broth (Difco TM B. D. Diagnostics various flow rates. This pressure drop was approximately Sparks Md.), which was supplemented with sodium chloride proportional to the flow rate. For each pressure drop measured to 3900 ppm and lactate to 2000 ppt, was used to enrich for at each flow rate, the base permeability of the sand pack was microbes that require more than a minimal medium for calculated. growth. The electronacceptor in the Marine broth sample was 0146 Pressure drop alone can be compared and used as a Scavenged from the Fe(III), Sulfate, nitrate and organic mol measure of the change in permeability between slim tubes ecules in the formulation that could be used as electronaccep since all the tubes had similar dimensions and received the tors. Each sample of medium was deoxygenated by sparging same flow rates of brine during the tests. the filled vials with a mixture of nitrogen and carbon dioxide 0147 The empty volume in the slim tubes, called the pore followed by autoclaving. All manipulations of bacteria were volume, was 40-50 ml. This pore volume was calculated from performed in an anaerobic chamber (Coy Laboratories Prod the product of the total volume of the slim tube and an esti ucts, Inc., Grass Lake, Mich.), and the cultures were incu mate of the porosity (about 40%). bated at ambient temperature.

Calculation of Base Permeability TABLE 4

0148. The base permeability of each tube was measured Minimal salts medium using the Brine #1 flowing at full pressure: about 95 psi (0.665 megapascal) in the slim tube (controlled at the outlet end with g/L Chemical the back pressure regulator) and about 110 psi (0.758 mega 1.O NHCl pascal) in the pressure vessel (outside of the slim tube). Base O.S KHPO. permeability was calculated using the Darcy Equation: 0.4 MgCl26H2O O.2 CaCL•2H2O 10 NaCl O.69 NaH2PO4 k = 4.08: Q: it: L 2.5 NaHCO A : AP US 2012/0277127 A1 Nov. 1, 2012 11

water. Seven isolates were named 97AE 3-12, 97AE3-1, TABLE 4-continued 97AE 3-3, 97AE 3-7, 97AE 4-6, 97AE 4-5, and 97AE4-1. Minimal salts medium Example 2 1000X g/L Trace elements Characterization of Isolated Strains with Respect to 1.5 FeCl24H2O Arcobacter sp. O.OO2 CuCl2.H2O O.1 MnCLP4H2O (O155 To determine the 16S rDNA sequence of the seven O.19 CoCl6HO isolates named 97AE 3-12, 97AE3-1, 97AE 3-3, 97AE 3-7, O.O7 ZnCl2 O.OO6 HBO 97AE 4-6,97AE 4-5, and 97 AE4-1 (Example 1), each of the O.036 Na-MoCl2.H2O seven isolates was picked as a pure single colony, DNA was O.O24 NiCl26H2O isolated and the 16S rRNA gene was amplified by PCR using O.277 HCI the procedures in General Methods. The amplified sequences were cloned into pCR-TOPO4 vector using the TOPO TA 1000X g/L Seleniumtungstate cloning system (Invitrogen), as recommended by the manu O.OO6 Na-SeOSHO facturer, and then sequenced multiple times using primers O.OO8 Na-WO2H2O 1492R, 8F, M13 Reverse, and M13 Forward of SEQID NOs: O.S NaOH 43-46, respectively, to obtain the near full sequence. Each 1000X mg/L Vitamin mix strain 16S rDNA sequence (97AE 3-12: SEQ ID NO:1, 97AE3-1: SEQID NO:33,97AE 3-3: SEQID NO:34,97AE 100 vitamin B12 8O p-aminobenzoic acid 3-7: SEQID NO:35,97AE 4-6: SEQ ID NO:38,97AE 4-5: 2O D(+)-Biotin SEQ ID NO:37, and 97 AE4-1: SEQID NO:36) was queried 200 nicotinic acid against the NCBI (National Center for Biotechnology Infor 100 calcium pantothenate mation) database using the BLAST (Basic Local Alignment 300 pyridoxine hydrochloride Search Tool) algorithm program provided by NCBI (Alts 200 thiamine-HCL•2HO chul, et al. (1990).J. Mol. Biol. 215:403-410) to identify the 50 Alpha-lipoic acid most similar nucleotide sequences. This was executed by comparing the query sequence to similar 16S rRNA 0151. The pH of the medium was adjusted to 7.3. sequences in the database and determining a score of relative 0152 The enrichments containing nitrate were monitored percent identity. All query sequences, one each for 97AE and sampled regularly for nitrate depletion and nitrite accu 3-12, 97 AE3-197AE 3-3, 97AE 3-7, 97AE 4-6, 97AE 4-5, mulation, or in Some cases, nitrite depletion. When nitrate and 97 AE4-1, returned top hits as Arcobacter marinus CL-S1 was depleted in the sample (usually by 14 days), lactate and (SEQ ID NO:4), Arcobacter sp. Solar Lake (SEQID NO:2), nitrate were added to the original final concentrations. Lactate or Arcobacter sp. YJO-18 (SEQID NO:3) at greater than or and the electron acceptor in each of the other enrichments equal to 99% identity. were added to the original final concentrations as well. In the 0156 Based on the initial Arcobacter identity, 24 16S marine broth enrichment sample, lactate was added for fur rDNA reference sequences in the NCBI database from the ther incubation. Arcobacter genus and 5 related sequences were selected. 0153 All vials were incubated for addition 14 to 20 days at These sequences are listed in Table 1 with SEQ ID NOs: room temperature. Changes which indicated growth on lac 2-6,8,9, and 11-31. Reference sequences included 10 from tate and the electron acceptor combination of each enrich Arcobacter strains (SEQ ID NOs:4, 6, 8, 9, 15, 17-21) that ment were observed in the vial samples. Changes included represent 12 different Arcobacter species (Type strains) rec visible turbidity in the medium, and the presence of biofilms ognized by the International Committee on Systematics of on the glass vials or at the gas-aqueous interface, as well as Prokaryotes (List of Prokaryotic names with Standing in nitrate and nitrite reduction in vials containing nitrate as the Nomenclature). Also the reference sequences included 6 electron acceptor. Turbidly was similar in each vial indicting strains from oil reservoirs (SEQID NOS:3, 5, 24, 25, 28 and that there was a diverse population of microorganisms in both 31). Other Arcobacter isolates and strains were included as the injection water and the production water. reference sequences because they had been referenced in peer 0154 After a second incubation of 14-20 days at room reviewed journals as Arcobacter sp. but had not yet been temperature, a 100 uL sample from each enrichment was critically typed and described as species (SEQ ID NOS:2. streaked onto Marine broth agar plates (made per recipe, 11-14, 16, 22, 23, 26, 27 and 30). In addition the E. coli K12 Difco 2216, Becton-Dickenson, Sparks, Md.) and incubated 16S rDNA B sequence (positions 8-1511; SEQ ID NO:32) at room temperature for two days. Representative colonies was used to serve as a scaffold for the sequence alignment and with unique morphologies were isolated, restreaked onto to provide the base coordinate system, which is recognized as Marine broth agar plates, and grown to purify isolates. the base position standard (Brosius, J., et al. (1981) J. of Samples of isolated colonies were screened for identification Molecular Biology, 148(2):107-127; Woese, (1987) Bacte by PCR amplification using direct colony rDNA analysis rial Evolution. Microbial Rev. 51: 221-271). The test described in the General Methods using both the reverse PCR sequence for alignment was from strain 97AE3-12 (SEQ ID primer 1492R (SEQ ID NO:43) and forward PCR primer NO:1). This sequence was representative of the other six 8F(SEQ ID NO:44). The DNA sequencing and analysis strains based on sequence stretches (500-700 bp) which described in General Methods was used to obtain 16S rRNA showed high sequence identity between all six strains and sequence for microbial identification. Isolates identified as Arcobacter marinus CL-S1 (GenBank: EU512920). belonging to Arcobacter were primarily obtained from the 0157. A global alignment was created using near full fumarate and marine broth enrichments of the production length 16S rDNA sequences of SEQ ID NOs: 1-6,8,9, and US 2012/0277127 A1 Nov. 1, 2012

11-32 with the Clustal W alignment algorithm (Chenna, R. (JCM 15502) (Kim, H. M. etal Int. J. Syst. Evol. Microbiol. H., et al., 2005 and Lark, M. A. etal, 2007). All 24 Arcobacter 60:53 (2010) which is the type reference strain with the high sequences aligned and showed a significant distance in iden est sequence identity at 99.5%. Other isolated and described tity to the E. coli K1216S rDNA with sequence identity of 76 strains in clade 1 include Arcobacter sp Solar Lake, Arco to 78%. All sequences demonstrated a 25 bp deletion when bacter halophilus LA31 B (ATCC BAA-10227) (Teske et al. compared to the E. coli K12 sequence in variable region 3 at (1996) Appl. Environ. Microbiol. 62:4210; Donachie et al base coordinate positions 452 to 476. There also was a 4 bp (2005) Int. J. Syst. Evol. Microbial. 55:1271). Though A. insertion (WGCT) in variable region 6 at base coordinate mytili F2075 and A. nitrofigillis DSM7299 have practically position 1028 and a 6 bp deletion in variable region 9 at base the same percent 16S rDNA sequence identity to 97AE3-12, coordinates 1451 to 1456. These structural signatures are the signature sequences of the 16S rDNA of these two strains consistent with the signature for the following phylogenetic place them in clades 1 and 2, respectively. classification: 0160 Phylogenetic clade 3 is anchored by Arcobacter but zleri (the most described and isolated Arcobacter species). Bacteria/Epsilonproteobacteria/Campylobacterales/ This clade also contains Arcobacter Skirrowi, Arcobacter Campylobacteraceae/Arcobacter. thereius, Arcobacter cibarius and Arcobacter Cryaerophilus. All of these strains are BSL2 organisms. 0158. The alignment yielded the closest sequence identi 0.161. In the same manner a molecular phylogenetic tree ties for strain 97AE3-12 to Arcobacter sp. sequences shown was generated to show the relatedness among newly isolated in Table 5. strains 97AE 3-12, 97 AE3-1, 97AE 3-3, 97AE 3-7, 97 AE 4-6, 97AE 4-5, and 97 AE4-1, and also their relationship to a TABLE 5 subset of the reference strains used above. This tree shown in 97AE3-12 and known 16S rDNA sequences FIG. 2 indicates the close relationship between the newly having closest sequence identities isolated Strains and the known strains Arcobacter sp. Solar Lake and Arcobacter sp. YJO-18 that are shown in FIG. 1 to Matching sequence description *Overlap Identity be in Arcobacter clade 1. Uncultured Arcobacter sp. clone 471,1473 99.9% 0162. Using the same global multiple sequence alignment YJO-18, 16S rRNA gene partial described above, signature positions in the 16S rDNA sequence. GenBank: AY569293.1 sequences were identified which may be used to distinguish Arcobacter sp. Solar Lake, Sinai 431,1437 99.7% Peninsula, 16S rRNA gene partial Arcobacter species in clade 1 from the Arcobacter species in sequence (GenBank: L42994.1) clades 2 and 3 by the signature sequences at these positions. Arcobacter marinus CL-S1 (JCM 419, 1426 99.5% These signature positions are listed in Table 6, with position 5502), 16S rRNA gene, partial sequence (GenBank: EU512920.2) coordinates from the E. coli 16S rDNA sequence. The con Bacterium enrichment culture clone 4321474 97.1% sensus sequence for Arcobacter sp. clade 1 at each of the EB27.1, 16S rRNA gene, partial signature positions is listed. At some positions a single nucle sequence. GenBank EU573100 otide occurs, while at other positions there is degeneracy Arcobacter halophilus LA31B 352.1402 96.4% (ATCC BAA-1022T), 16S rRNA gene, where R may be A or G.Y may be C or T. M. may be A or C. partial sequence, GenBank: AF513455.1 K may be G or T. S. may be C or G, W may be A or T. B may Arcobacter mytili strain F2075 3341401 94.5% be C, G, or T. D may be A, G, or T H may be A, C, or TV may (LMG 24559), 16S rRNA gene, be A, C, or G and N is A, CG or T. partial sequence GenBank: EU669904.1 Arcobacter nitrofigilis (DSM 7299), 337,1477 94.6% 0163. In Table 6 the Arcobacter sp. clade 1 consensus complete genome, GenBank: CP001999.1 nucleotides at each signature position are compared to the Arcobacter butzieri RM4018, complete 375.1479 93.0% consensus nucleotides for each signature position of Arco genome. GenBank: AY570593 Bacterium enrichment culture clone 366,1477 92.5% bacter sp. clade 2 and clade 3. In addition to consensus PL-8B1, 16S rRNA gene, partial sequence. nucleotides of clade 1, the nucleotides present at each signa Low temperature oil reservoir GenBank ture position in clade 1 strains 97AE3-12, Arcobacter mari Thiomicrospira sp. CVO, 16S O88, 1277 85.2% nus CL-S1 and Arcobacter halophilus LA31B are shown in rRNA gene, partial sequence Table 6. The nucleotides at all of the signature positions GenBank: U46506.2 together, for each of these strains, identifies these strains as Overlap means the length of overlapping sequence that was used to determine the percent Arcobacter sp. clade 1, while there are differences from the identity (includes mismatches, deletions, and insertions betweenfixed ends). This varies due to variability of available sequence length for different 16S rDNA sequences. Arcobacter sp. clade 1 consensus nucleotides among the sig nature positions for Arcobacter sp clade 2 and clade 3 species. 0159. A phylogenetic tree was created by ClustalWalign 0164. The majority of the signature positions identified ment with the same rDNA sequences (SEQID NOs: 1-6,8,9, were located in the hypervariable regions of the 16S rDNA, 11-32) using the phylogenetic tree and bootstrapping func with positions designated by nucleotides of the 16S rDNA tions of the MegAlignTM program in the DNAStar LaserGene sequence from E. coli: package (LaserGeneTM DNASTAR, Inc Madison, Wis.). The 0.165 hypervariable region 1 between positions 44 and phylogenetic tree shown in FIG. 1 shows all Arcobacter ref 110 erence strains with strain 97AE3-12. The ten recognized Arcobacter reference strains form three clades (1, 2, and 3) in 0166 hypervariable region 2 between positions 120 and the molecular phylogenetic analysis, A. marinus and A. halo 3OO philus are in clade 1. A. nitrofigillis is in clade 2, and the 0.167 hypervariable region 3 between positions 365 and known pathogens represented by A. butzlerii are all inclade 3. 500 As shown in FIG. 1, strain 97 AE3-12 falls within the Arco 0168 hypervariable region 4 between positions 574 and bacter clade 1 which includes Arcobacter marinas CL-S1 750 US 2012/0277127 A1 Nov. 1, 2012 13

0169 hypervariable region 5 between positions 820 and dominant consensus 16S rRNA sequence for Arcobacter sp. 88O clade 1 16S rDNA is SEQ ID NO:39. 16S rDNA sequences 0170 hypervariable region 6 between positions 990 and containing all of the signature sequences in Table 6 for Arco 1050 bacter clades 2 and 3 are SEQ ID NOs:41 and 42, respec 0171 hypervariable region 7 between positions 1115 and tively. An alignment of the 16S rDNA sequences for Arco 1175 bacter clade 1 dominant consensus, Arcobacter clade 1 0172 hypervariable region 8 between positions 1240 and degenerate consensus, strain 97AE3-12, Arcobacter clade 2 1370 degenerate consensus, and Arcobacter clade 3 degenerate 0173 hypervariable region 9 between positions 1415 and consensus is shown in FIG. 3. The differences between the 1465 16S rDNA signature sequences for clades 1, 2, and 3 are in 0.174. The identified signature sequences in the 16S rDNA bold and underlined. Explanations for the notations in FIG. sequence may be used to identify microorganism strains as 3A-D: belonging to Arcobacter clade 1 as opposed to Arcobacter sp. (0175 No bracket around “N” or “-” (insertion ordeletion, clade 2 or 3. A composite degenerate 16S rDNA sequence for respectively) means that is the dominant and only sequence (it Arcobacter sp. clade 1 that contains all of the degenerate may be degenerate as indicated by the letter used). signature sequences in Table 6 is SEQID NO:40. This degen- (0176 NI means the nucleotide exists in more than one erate sequence includes insertion/deletion positions in Table reference sequence; it is prevalent but also a '-' exists in our 6. Known or newly isolated microbial strains may be identi- isolates and nearest neighbors. fied as belonging to Arcobacter sp. clade 1 by having 16S 0177 (N) means the nucleotide only exists in one refer rDNA that is of SEQ ID NO:40. The most prevalent, or ence sequence and is dominated by a '-' at that position.

TABLE 6 16S rDNA signature sequences for distinguishing Arcobacter Clade 1 from Arcobacter Clades 2 and 3 including nucleodtides for Arcobacter Clade 1 dominant and degenerate consensus signature sequences and for strains 97AE3 - 12, Arcobacter marinus, and Arcobacter halophilus at the signature postitions using coordinates of E. coli 16S rDNA

Aircobacter Arcobacter Arcobacter Aircobacter E. coli K12 Clade 1 Clade 1 Clade 2 BSL1 Clade 3 BSL2 W311 O ra Dominant Degenerate Degenerate Degenerate Coordinate Arcobacter Arcobacter Consensus Consensus Consensus Consensus No. 97AE3 - 12 marinus halophilus Signature Signature Signature Signature

44 - 48 GTGCT GTGCT GTGCT GTGCT GTGCT GTGCT GTGCT

69-70 AG AG AG AG AG AG AG

79 1 nt k 1 n. t. A. A. 1 n. t. A. A. deletion deletion deletion or A.

73-83 CGGGAT-- CGGGAT-- CGGATTATAG CGGGATATA CGGRWTATA CGGRTTAWA CGGATTATAG TAGC TAGC C GC GC GC C

89 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. insertion insertion T insertion insertion T insertion T T or no insertion

86-93 GCTAATCT GCTAATCT GCTATAATT GCTA (T) AATT GCTA (T) AWYT GCTW (W) WW GCTA (T) ARTT YT

1 OO 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. insertion T insertion T insertion T insertion T insertion T insertion T insertion T

94 - 1 OO GTCAGCTA GTCAGCTA GTCAGCTA GTCAGCTA GTCAGCTA GTCAGCTA GTCAGCTA

108 C C C C C C C

122-126 ATATA ATATA ATATA ATATA RTATA RTATA RTATA

132 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. M A. insertion G insertion G insertion G insertion G insertion R

128-133 GTAACGT GTAACGT GTAACGT GTAACGT GTAACRT GTAACMT GTAATAT

137-149 TTACAAGAGG TTCAAGAGG TCTAAGAGGG TTCAAGAGG YYYAAGAGGG CTMDAGARR TCTTACTAAG GGGA GGGA GGA GGGA GGA RGRA GGA

154 - 156 AGA AGA AGA AGA AGW AGW ARW

165 - 167 TCT TCT TCT TCT WCT WCT WYT US 2012/0277127 A1 Nov. 1, 2012 14

TABLE - Continued 16S rDNA signature sequences for distinguishing Aircobacter Clade 1 from Arcobacter Clades 2 and 3 including nucleodtides for Arcobacter Clade 1 dominant and degenerate consensus signature sequences and for strains 97AE3 - 12, Arcobacter marinus, and Arcobacter halophilus at the signature postitions using coordinates of E. coli 16S rDNA

Arcobacter Aircobacter Aircobacter Arcobacter E. coli K12 Clade 1 Clade 1 Clade 2 BSL1 Clade 3 BSL2 W311 O ra Dominant Degenerate Degenerate Degenerate Coordinate Aircobacter Arcobacter Consensus Consensus Consensus Consensus No. 97AE3 - 12 marinus halophilus Signature Signature Signature Signature

173 - 178 AACCCC GACCCC TACCTT AACCCC DRYCYY CACCCC TACC TT

182-188 TGCCTTT TGCCTTT TGCCTTT TGcc TTT TGCCTTT TGCCTTT YTCCWYY

189-2O4. AATGCGAAA AATACAAAA AATACGAAAGT AATACGAAA AATRCDAAAGT AAKACHYAW YYAWCHWAA GTATGCA GTATGAA ATGCA GTATGCA ATGMA GTYTGCA GWTRRWA

211 1 n. t. 1 n. t. insertion C insertion C or no insertion

2O6-219 GGGAAATAT GGGAAATAT GGGAAACGCT GGGAAATAT GGGAAA (C) KY GGGAAACAT GGGAAAGATT TTATA TTATA TTAGT TTATA TTWAKW TTATG TATT

221-226- CTTGAA CTTGAA CTTAGA CTTGAA CTTRRR CTCTAG GTAAGA 23 O-233 CGGC CGGC CGGC CGGC TGGC KGGY TAGC

235-242 TGTACAGT TGTACAGT TGTACAGT TGTACAGT TGTAYWGT TGTAYRGT TGTATTGT

245 C C C C C C C

248 - 25 O ATA ATA CTA ATA MTA MTM TTA

253 T T T T T T T

257-59 GAG GAG GAG GAG GRG GAG GGG

264 - 265 TA TA TA TA KA TG TG

267-269 CTC CTC CTC CTC CYY CTC CCT

273 A. A. A. A. A. A. A.

276-28O TCAAT TCAAT GCAAT TCAAT DCAAT RCAAT ACDAT

283 - 288 CGCTTAA CGCTTAA CGCTTAA CGCTTAA CGCWTAA CRCYTAA CGCATAA

295 T T T T T T T

3. Of T T T T T T T

311 T T T T T T T

378-385 GGGAAACC GGAAACC GGGAAACC GGGAAACC GGGRAACC ACGAAAGT ACGAAAGT

396-398 AAC AAC AAC AAC AAC AAC AAC

407-419 GAGGATGAC GAGGATGAC GAGGATGACA GAGGATGAC GAGGATGACA GAGGATGAC GAGGATGACA ACAT ACAT CAT ACAT CAT ACAT CAT

425 - 427 TGC TGC TGC TGC TGC TGC TGC

433 - 435 CTC CTC CTC CTC CTC CTC CTC

440-449 TATATAAGAA. TATATAAGAA TATATAGGAA TATATAAGAA TATATARGAA TATATAGGAA TATATAAGAA

452- 479 25 nt. 25 nt. 25 nt. 25 nt. 25 nt. 25 nt. 25 nt. deletion -- deletion -- deletion - deletion - - deletion - - deletion - - deletion - -

477 - 479 TAA TAA AAA TAA WAA TAA TAA

484 - 497 GGTATTATAT GGTATTATAT GGTACTATATG GGTATTATAT GGTAYTATATG GGTACYATA GGTATTATAT GAAT GAAT AAT GAAT AAT TGAAT GAAT US 2012/0277127 A1 Nov. 1, 2012 15

TABLE - Continued 16S rDNA signature sequences for distinguishing Aircobacter Clade 1 from Arcobacter Clades 2 and 3 including nucleodtides for Arcobacter Clade 1 dominant and degenerate consensus signature sequences and for strains 97AE3 - 12, Arcobacter marinus, and Arcobacter halophilus at the signature postitions using coordinates of E. coli 16S rDNA

Arcobacter Aircobacter Aircobacter Arcobacter E. coli K12 Clade 1 Clade 1 Clade 2 BSL1 Clade 3 BSL2 W311 O ra Dominant Degenerate Degenerate Degenerate Coordinate Aircobacter Arcobacter Consensus Consensus Consensus Consensus No. 97AE3 - 12 marinus halophilus Signature Signature Signature Signature

5O2 A. A. G A. R A. A.

539 A. A. A. A. A. A. R

543 T T C T Y T T

554 C C C C C C C

562 C C C C C Y C

576-582 AGCGTGT AGCGTGT AGCGTGT AGCGTGT AGCGTGT AGCRTGT AGCRTGT

589 - 5.93 ATAGA ATAGA ATAGA ATAGA ATMRA GTAWW ATTRA

599 - 603 CAGAA CAGAA TAGGA CAGAA YAGRA. YDGAA TTGAA

613 - 62O AATAGCTT AATAGCTT TATGGCTC AATAGCTT WATRGCTY WATRGCTY TATAGCTT

624 - 627 TATT TATT CATA TATT YATW YATW TATA

635 - 6 41 TTTTGAA TTTTGAA TTCTAA TTTTGAA TTYTRAA TTCCAAA TTTGAAA

64 6- 658 TCTATCTAGA TCTATCTAGA TCTATCTAGAG TCTATCTAGA TYWATCTAGA KTAACCTAG TTAACCTAGA GTA GTA TA GTA GTA AATR ATG

7Os G G G G G K G

734 - 738 ATCTA ATCTA ATCTA ATCTA ATCTA ATCTA ATCTA

744 - 75.1 ACATAACT ACATAACT ACATAACT ACATAACT ACATAACT ACAYWATT ACAHTATT

758 - 764 GAGACGC GAGACGC GAGACGC GAGACGC GAGACGC GAGAYGC GAGAYGC

822-824 TAC TAC TAC TAC TAC YAC TAC

835-84 O GCTATG GCTATG GCCATG GCTATG GCYATR GTGAGG GTGAGR

842 - 848 CGACATA CGACATA CGACATG CGACATA CGACATD AGACCTT YGAYCTT

878 A. A. A. A. A. R A.

903 A. A. A. A. R A. A.

948 C C C C Y C C

971. G G G G G R A.

989 T T T T T W A.

998 - 10 O2 AGTAA AGTAA AGTAA AGTAA AGWAA AGWAA AGTAA

1 OO6-1011 CCATTT CCATTT CCATTT CCATTT YHMTYY CNTWHY YKWTYW

1027-1028 TC TC TC TC YY YY TC

1028s 4 n. t. 4 n. t. 4 n. t. 4 n. t. 4 n. t. 4 n. t. 4 n. t. insertion insertion insertion insertion insertion insertion insertion TGCT TGCT TGCT TGCT WGCT WGCT TGCT

103 O-1034 GCAGA GCAGA GCAGA GCAGA GCWRR GCWRR GCAGA

1036 A. A. G A. R R A.

1038 - 1043 TTATAT TTATAT TTATAT TTATAT TTWYAT TWWYAT TTRYAT

US 2012/0277127 A1 Nov. 1, 2012 17

TABLE 6 - continued 16S rDNA signature sequences for distinguishing Aircobacter Clade 1 from Arcobacter Clades 2 and 3, including nucleodtides for Arcobacter Clade 1 dominant and degenerate consensus signature sequences and for strains 97AE3 - 12, Arcobacter marinus, and Arcobacter halophilus at the signature postitions using coordinates of E. coli 16S rDNA

Arcobacter Aircobacter Aircobacter Arcobacter E. coli K12 Clade 1 Clade 1 Clade 2 BSL1 Clade 3 BSL2 W311 O ra Dominant Degenerate Degenerate Degenerate Coordinate Aircobacter Arcobacter Consensus Consensus Consensus Consensus No. 97AE3 - 12 marinus halophilus Signature Signature Signature Signature

361 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. 1 n. t. insertion insertion insertion insertion insertion insertion insertion sC& sC& sC& sC& sC& sC& sC&

362 A. A. A. A. W W T

364 T T T T Y T T

367 T T T T T W T

37 O 1 n. t. insertion (A)

396 T T T T W T T

419 G G G G G G G

1421-1426 TGATTT TGATTT TGATTT TGATTT TGAWTT TGANYT TGAACT

1428-1332 ACTCG ACTCG ACTCG ACTCG ACYCG ATTCG ATTCG

1436-144 O CGGGG CGGGG CGGGG CGGGG CRGGG CGGRG CGGGG

1444 C C C C Y C C

1448- 1449 GA GA GA GA RR AR AR

1451 - 1456 6 n. t. 6 n. t. 6 n. t. 6 n. t. 6 n. t. 6 n. t. 6 n. t. deletion -- deletion -- deletion - - deletion - - deletion - - deletion - - deletion - -

1458 G G G G R G G

1464 C C C C Y Y T

1468 A. A. A. A. H A. A.

1475 A. A. A. A. W W T

1477 T T T T Y Y Y

1503 A. A. A. A. R R A.

R = AAG; K = G/T; S = C/G; Y = c/T; M = A? C; W = AAT; D = A? GAT not C; H = A? CAT not G; B = CAGAT not A; W = A? CAG not T n. t. is nucleotide

Example 3 another. As a reference strain, Arcobacter halophilus (ATCC strain BAA-1022), which has 16S rDNA with 96.4% Riboprinting to Determine Strain Differences sequence identity to strain 97AE3-12, was included in the 0.178 The Arcobacter sp. strains isolated in Example 1: analysis. While all of these strains have significant sequence 97AE3-12,97AE3-197AE 3-3, 97AE 3-7,97AE4-6,97AE identity to one another in the 16S rDNA, several unique 4-5, and 97AE4-1 were subjected to automated Riboprinter(R) rDNA RiboPrintTM patterns were obtained. As shown in FIG. analysis, as described in General Methods, to determine 4, the patterns of EcoRI restriction fragments which hybrid whether these isolated Strains were unique with respect to one ized to 16S and 23S rDNA probes were different for Arco US 2012/0277127 A1 Nov. 1, 2012

bacter sp. representative strains 97AE3-12 (ATCC #PTA 97AE3-12 inoculum were prepared in duplicate. A single 11409), 97AE3-3 (ATCC #PTA-11410), and 97 AE3-1. This non-inoculated control (NIC) per test medium was prepared analysis showed that the genomic sequences Surrounding the as an abiotic control. Nitrate reduction was analyzed as a 16S and 23 rRNA genes in these strains are different from one measurement of cell growth in each medium by IC as another and also are different from the tested comparator described in General Methods. 97AE3-12 reduced 100% of strain Arcobacter halophilus. the nitrate provided to nitrite within 6 days of incubation at 25° C. in low salt medium supplemented with lactate, and Example 4 reduced 50-77% of the nitrate to nitrogen in the high salt Screening of Bacterial Isolate 97AE3-12 for Growth medium supplemented with lactate. Results in Table 7 show in the Presence of Oil Under Low and High Salt that Arcobacter sp. strain 97AE3-12 was able to grow using Conditions lactate as a carbon source in the presence of petroleum from the Wainwright Well, in both a low and a high salts medium. (0179 Cultures of strain 97AE3-12 were grown anaerobi cally in the presence of oil using both a low salt minimal salts TABLE 7 medium and a high Salt synthetic brine formulation. The high salt synthetic brine used had a salinity of 64 ppt, which is Nitrate reduction in cultures of 97AE3-12 in the about two times the salinity of sea water: The high salt syn presence of oil, in low or high Salt media thetic brine was composed of the following: NaCl, 55.0 g/L, % Nitrate Reduction NHCl, 0.1 g/L, KHPO, 0.05 g/L. NaSO, 0.1 g/L, selen ite-tungstate solution NaOH, 0.5g/L, Na-SeO.5H2O, 6.0 Day: 1 2 6 17 29 mg/L. NaWO.2H2O, 8.0 mg/L), 1 mL/L. NaHCO, 0.2 g/L, Test1: low salt plus 32.7% 75.0% 100.0%. 100.0% 98.1% vitamin solution Vitamin B12, 100 mg/L, p-aminobenzoic actate acid, 80 mg/L, D(+)-Biotin, 20 mg/L. Nicotinic acid, 200 NIC low salt-plus 23.6% -4.2% -3.9% O.0% -1.6% mg/L, Calcium pantothenate, 100 mg/L, Pyridoxine hydro actate Test2: low salt no 12.1% 6.3% 11.5% O.0%. 22.9% chloride, 300 mg/L. Thiamine-HC1.2 H2O, 200 mg/L, Alpha actate lipoic acid, 50 mg/L), 1 mL/L, SL-10 trace metal solution NIC-low salt no 14.0% -2.2% -1.4% O.0% 9.6% 25% HCl, 10 mL/L. FeC14H2O, 1.50 g/L, ZnCl2, 70 mg/L, actate MnCl2.4H2O, 100 mg/L, HBO, 6 mg/L, CoCl2.6H2O, 190 Test3: high salt plus 11.2% 32.4% 77.0% SO.3% 62.2% actate mg/L, CuCl2 HO, 2 mg/L. NiCl6 HO, 24 mg/L, NIC-high salt plus 11.6% -1.7% -O.8% O.0%. 13.9% NaMoO2 H2O, 36 mg/L), 1 mL/L, CaCl2 H2O, 8.8 g/L, actate NaNO, 2.0 g/L, KC1, 0.86 g/L, MgCl2.6 HO, 6.4 g/L, and Test4: high Salt no 11.7% 6.1% -15.6% O.0%. 27.8% supplemented with NaNO, 2.0 g/L, pH 6.7 and 64 ppt salin actate NIC-high salt no 16.4% 10.8% -21.8% O.0%. 30.9% ity. actate 0180. The low salt medium was composed of NaCl, 10 g/L, NHCl, 1.0 g/L, KHPO, 0.5g/L, KSO, 0.1 g/L, selen ite-tungstate solution NaOH, 0.5g/L, NaSeO.5 HO, 6.0 mg/L. NaWO.2H2O, 8.0 mg/L), 1 mL/L. NaHCO, 2.5g/L, Example 5 vitamin solution Vitamin B12, 100 mg/L, p-aminobenzoic acid, 80 mg/L, D(+)-Biotin, 20 mg/L. Nicotinic acid, 200 Screening Bacterial Isolates for their Ability to Form mg/L, Calcium pantothenate, 100 mg/L, Pyridoxine hydro Biofilms and Plug Flow chloride, 300 mg/L. Thiamine-HC1.2 H.O. 200 mg/L, Alpha lipoic acid, 50 mg/L), 1 mL/L, SL-10 trace metal solution 0182 Arcobacter strains 97AE 3-12,97AE3-197AE 3-3, 25% HCl, 10 mL/L. FeC14H2O, 1.50 g/L, ZnCl2, 70 mg/L, 97AE 4-6, 97AE 4-5, 97AE 3-7, and 97AE4-1, that were MnCl2.4H2O, 100 mg/L, HBO, 6 mg/L, CoCl2.6H2O, 190 isolated from a Canadian oil reservoir as described in mg/L, CuCl2.2 H2O, 2 mg/L. NiCl2.6 H2O, 24 mg/L. Example 1 above, were tested for their ability form biofilms to NaMoO2 HO, 36 mg/L), 1 mL/L, CaCl2 HO, 0.1 g/L, inhibit flow in medium porosity (median pore diameter=10 MgCl2.6H2O, 0.2 g/L, yeast extract, 0.5g/L, and NaNO, 2.0 microns) sintered glass filters as described in General Meth g/L, pH 6.9 and 15 ppt salinity. ods. The high Salt medium used for testing had the following 0181. Both low and high salt media were tested for their composition: NaCl, 54 g/L, NHCl, 0.1 g/L, KHPO, 0.05 ability to support growth of Arcobacter sp. 97AE3-12 with g/L. NaSO, 0.1 g/L, selenite-tungstate solution NaOH, 0.5 and without sodium lactate added as a carbon source at about g/L, Na-SeO.5H2O, 6.0 mg/L. NaWO2 H2O, 8.0 mg/L). 1 g/L (sodium lactate 60% syrup, 1.3 ml/L). Sodium nitrate was added at 2 g/L in media as the electronacceptor for all test 1 mL/L. NaHCO, 0.2 g/L, vitamin solution Vitamin B12, samples. Media was degassed and 12 mL added to 20 mL 100 mg/L. p-Aminobenzoic acid, 80 mg/L, D(+)-Biotin, 20 serum vials. 6.0 mL of degassed autoclaved petroleum oil mg/L. Nicotinic acid, 200 mg/L, Calcium pantothenate, 100 from Well #2 in the Wainwright field in the province of mg/L, Pyridoxine hydrochloride, 300 mg/L. Thiamine-HC1.2 Alberta, Canada was added to each vial. This oil was the sole H2O, 200 mg/L, Alpha-lipoic acid, 50 mg/L), 1 mL/L, SL-10 carbon Source in the samples lacking lactate. 0.45 ml of trace metal solution 25% HCl, 10 mL/L. FeC14H2O, 1.50 97AE3-12 grown in an undefined medium (Sea salts (Sigma g/L, ZnCl, 70 mg/L, MnO14 H.O. 100 mg/L, HBO, 6 S9883), 60.0 g/L, Peptone 5.0 g/L, Yeast Extract 5.0 g/L mg/L, CoCl2.6 HO, 190 mg/L, CuCl2 H2O, 2 mg/L, Casamino Acids 5.0 g/L. NaFeEDTA 133 mg/L, with pH NiC1.6 HO, 24 mg/L, Na MoO2 H2O, 36 mg/L. 1 mL/L, adjusted to between 6.4-6.6 and filter sterilized) was added as CaCl2 HO, 8.8 g/L, KC1, 0.86 g/L, MgCl,.6 HO, 6.4 g/L, inoculum to the anaerobic test cultures to about 5x107 cells of disodium fumarate, 3275 g/L, sodium lactate, 1 g/L. This inoculum per ml of medium. Test cultures which contained medium has salinity of 75 ppt. US 2012/0277127 A1 Nov. 1, 2012

0183 Each Arcobacter strain was inoculated into medium Example 6 of the above composition and incubated aerobically for 48 h. To initiate the test run 1 mL of a 48 h culture was added to 25 97AE3-12 Arcobacter Strain Biofilm Assay in Low mL of the same medium in triplicate. Triplicate filter assem and High Salt Media with Lactate as Carbon Source blies containing the inoculated medium were individually sealed in 125 mL incubation vessels under anaerobic condi 0185. Strain 97AE3-12 was assayed for the ability to form tions and placed in an incubator/shaker at 28°C. at 100 rpm biofilms on sintered glass filters as described in General for 2 weeks. In addition triplicate, uninoculated controls with Methods using three different media ranging in Salinity from the same medium formulation, but without the strain inocu 15 ppt to 68 ppt. Salinity of each media was measured by lum were run in parallel with the inoculated test samples. refractometer. 97AE3-12 was grown anaerobically in growth 0184. After two weeks, flow rates were checked by pass media of the following compositions: ing 1 mL of deionized water, gravity driven, through the 0186 Medium 1: minimal salts medium; NaCl, 10 g/L, filters. Time for water passage was measured three times in NHCl, 1.0 g/L. KHPO, 0.5g/L, KSO, 0.1 g/L, selenite succession for each of the test and control filters. Flow rates tungstate solution NaOH, 0.5 g/L, Na-SeO.5 H2O, 6.0 were calculated and post incubation values were compared to mg/L. NaWO.2H2O, 8.0 mg/L), 1 mL/L. NaHCO, 2.5g/L, preincubation values for each filter. Results in Table 8 show vitamin solution Vitamin B12, 100 mg/L, p-aminobenzoic that all strains tested caused a significant decrease in flow rate acid, 80 mg/L, DH-Biotin, 20 mg/L. Nicotinic acid, 200 versus the controls, which had an average of 27% increase in mg/L, Calcium pantothenate, 100 mg/L, Pyridoxine hydro flow rate. The increased flow rate resulted from better water chloride, 300 mg/L. Thiamine-HC1.2 H2O, 200 mg/L, Alpha saturation of the frit pores after two weeks of submersed lipoic acid, 50 mg/L), 1 mL/L, SL-10 trace metal solution incubation. These results demonstrate the capability of the 25% HCl, 10 mL/L. FeC14HO, 1.50 g/L, ZnC1, 70 mg/L, isolated Arcobacter strains to form biofilms and plugpores in MnCl2.4H2O, 100 mg/L, HBO, 6 mg/L, CoCl2.6H2O, 190 sand. mg/L, CuCl2.2 H2O, 2 mg/L. NiCl2.6 H2O, 24 mg/L. NaMoO2 HO, 36 mg/L), 1 mL/L, CaCl2.H2O, 0.1 g/L, TABLE 8 MgCl2.6H2O, 0.2 g/L, yeast extract, 0.025 g/L, NaNO, 2.0 g/L, sodium lactate 60% syrup, 1.3 ml/L, Bromothymol blue Changes in flow rate through medium porosity glass filters after solution, 0.4%, 3 mL. The salinity of this medium is 15 ppt. two week incubation with Arcobacter isolates 0187 Medium 2 equals Medium 1 in composition but with NaCl increased to 30g/L. The salinity of this medium is 35 flow, ml/sec ppt. 0188 Medium 3 is a high salts medium which includes pre Mean 96 higher levels of NaCl and the cations Ca++ and Mg++: NaCl, incu- post incubation % change 51.5 g/L, NHCl, 0.1 g/L, KHPO, 0.05 g/L NaSO, 0.1 bation values change in in flow g/L, selenite-tungstate solution NaOH, 0.5g/L, Na-SeO.5 treatinent value #1 #2 #3 mean flow rate' rate H2O, 6.0 mg/L. NaWO2 H2O, 8.0 mg/L. 1 mL/L, NaHCO, 0.2 g/L, vitamin solution Vitamin B12, 100 mg/L, control 1 O.077 O.091 0.100 0.100 0.097 +26 +27 p-aminobenzoic acid, 80 mg/L, DH-Biotin, 20 mg/L. Nico control 2 O.083 0.1OO 0.100 0.1OO O.1OO +21 tinic acid, 200 mg/L, Calcium pantothenate, 100 mg/L, Pyri control 3 O.083 0.111 0.111 O.111 0.111 +34 doxine hydrochloride, 300 mg/L. Thiamine-HC1.2 H2O, 200 97AE 3-12 OO67 O.O29 O.O29 O.O31 O.O29 -57 -56 mg/L. Alpha-lipoic acid, 50 mg/L, 1 mL/L, SL-10 trace Replicate O.O63 0.026 O.O29 O.O29 O.O28 -56 metal solution 25% HCl, 10 mL/L. FeC14H2O, 1.50 g/L, Replicate O.O63 0.028 O.O29 O.O31 O.O29 -54 ZnCl2, 70 mg/L, MnCl4 H2O, 100 mg/L, HBO, 6 mg/L, 97AE 3-3 O.111 O.083 0.083 0.083 0.083 -25 -48 CoCl6 HO, 190 mg/L, CuCl2 H2O, 2 mg/L. NiC1.6 Replicate O-111 O.O4O 0.043 O.045 0.043 -61 H2O, 24 mg/L. NaMoO.2 H2O, 36 mg/L. 1 mL/L, CaCl2 Replicate O.143 O.OS6 O.OS9 O.OS9 O.OS8 -59 H2O, 8.8 g/L, yeast extract, 0.025 g/L, NaNO, 2.0 g/L, 97AE 4-6 O.143 O.O71 O.077 O.O71 O.O73 -49 -52 sodium lactate 60% syrup, 1.3 ml/L, KC1, 0.86g/L, MgCl2.6 Replicate O.O71 O.O31 O.O33 0.036 O.O33 -54 H2O, 6.4 g/L, Bromothymol blue solution, 0.4%, 1 mL. The Replicate O.OS3 O.O24 O.O26 O.O27 O.O2S -53 97AE 4-5 O.100 OO37 0.042 0.042 0.040 -60 -61 salinity of this medium is 68 ppt. Replicate O.125 0.042 0.045 0.048 0.045 -64 0189 The experiment and flow rate tests after 2 weeks of Replicate O.O63 O.O2S O.O27 0.026 O.O26 -59 incubation were performed as described in General Methods. 97AE 3-7 OO67 0.027 O.O29 O.O31 O.O29 -57 -57 Medium inoculated with Arcobacter sp. 97AE3-12 were pre Replicate O.091 O.O33 O.O38 0.038 0.037 -59 pared in triplicate; non inoculated controls were prepared in Replicate O.OSO O.O22 O.O22 O.O23 O.O22 -56 duplicate. While the flow rate increased in the controls as in 97AE 4-1 O.091 O.OS9 O.OS9 O.O67 0.061 -33 -50 Example 5, strain 97AE3-12 caused a significant decrease in Replicate O.111 O.O37 O.O38 0.04O O.O38 -66 flow rate (Table 9). The flow rates in the control treatments Replicate increased by an average of 54, 35 and 46% for Medium 1, 2 and 3, respectively. The test treatments containing the 3 successive measurements replicate, 97AE3-12 inoculum showed a mean decline of 80, 80 and 'calculated as ((mean post incubation, mL secpreincubation, mL sec) - 1) x 100 46% in flow rate for medium 1, 2 and 3, respectively which had salinities of 15, 35 and 68 ppt, respectively. US 2012/0277127 A1 Nov. 1, 2012 20

TABLE 9 Changes in flow rate through medium porosity glass filters after two weeks incubation. post incubation pre- flow flow flow Mean % incubation rate, rate, rate, flow change % change Mean flow ml/sec ml/sec ml sec rate, in flow in mean rate, mlsec 1 2 3 ml sec rate' flow rate test1 15 ppt O-111 O.O17 O.O18 O.O17 O.O17 -84 -80 test2 15 ppt O.100 O.O2O O.O20 O.O2O O.O2O -80 test3 15 ppt O.091 O.O22 O.O22 O.O22 O.O22 -76 control1 15 ppt O.143 O.2OO O.200 O.167 0.189 32 S4 control.2 15 ppt O.143 O.2SO O2SO O.2SO O.2SO 75 test1 35 ppt O.143 O.O3O O.O31 O.O31 O.O31 -78 -80 test235 ppt O.143 O.O27 O.O24 O.O24 O.O2S -82 test3 35 ppt O.12S O.O26 O.O27 O.O28 O.O27 -78 controll 35 ppt O.100 O.12S O-111 O.12S O.120 2O 35 control.235 ppt O-111 O.167 0.167 O.167 0.167 50 test1 68 ppt O.091 O.O67 O.O71 0.077 O.O72 -21 -46 test1 68 ppt O.12S O.OS6 O.OS6 0.059 O.057 -SS test1 68 ppt O-111 O.043 O.043 O.O38 O.042 -62 controll 68 ppt O-111 O.2OO O.200 O.2OO O.200 8O 46 control.268 ppt O-111 O.12S O.12S O.12S O.12S 13

3 successive measurements 'calculated as ((mean post incubation, mL secpreincubation, mL sec) - 1) x 100

Example 7 cate control tubes were not inoculated. Tubes were statically incubated for 7 days at 30° C. After seven days the mean Aggregation of Silica Particles OD600 of the duplicate, inoculated tubes and duplicate uni 0.190 Arcobacter strain 97 AE3-12 was tested for its abil noculated control tubes was about 0.04. When treatment ity to aggregate grains of crystalline silica as described in tubes were mixed vigorously by 10 seconds of Vortexing, General Methods. The medium used for testing had the fol turbidity increased dramatically due to resuspension of the lowing composition: NaCl, 54 g/L, NHCl, 0.1 g/L, KHPO, crystalline silica, which had settled to the tube bottoms. The 0.05 g/L, Na2SO, 0.1 g/L, selenite-tungstate Solution decline in turbidity due to settling of the crystalline silica was NaOH, 0.5g/L, Na-SeO.5HO, 6.0 mg/L, NaWO2HO, monitored over time after mixing by measuring OD600. 8.0 mg/L), 0.5 mL/L NaHCO, 0.1 g/L, vitamin solution Vitamin B12, 100 mg/L. p-Aminobenzoic acid, 80 mg/L, Results in Table 10 showed that turbidity declined much more D(+)-Biotin, 20.00 mg/L. Nicotinic acid, 200 mg/L, Calcium rapidly in the inoculated treatments than in the controls as pantothenate, 100 mg/L, Pyridoxine hydrochloride, 300 indicated by the percent reduction in OD600 for the inocu mg/L. Thiamine-HC1.2 H2O, 200 mg/L, Alpha-lipoic acid, 50 lated culture vs the control at 1 min and 10 min after mixing. mg/L. 1.0 mL/L, SL-10 trace metal solution 25% HCl, 10 0.192 This resulted from the silica particles forming large mL/L. FeCl4 HO, 1.50 g/L, ZnCl2, 70 mg/L, MnO1.4 clumps, up to 100 microns in diameter as determined by H2O, 100 mg/L, HBO, 6 mg/L, CoCl2.6 H2O, 190 mg/L, microscopic examination, in the inoculated treatments, which CuCl2 HO, 2 mg/L. NiC1.6 HO, 24 mg/L. NaMoO.2 settled rapidly compared to the dispersed, unaggregated, 2-20 H2O, 36 mg/L), 1.0 mL/L, CaCl2.H2O, 4.4 g/L, 0.25g yeast p particles in the uninoculated control tubes. The contrasting extract, 0.5 g casein peptone, KCl, 0.86 g/L, MgCl2.6 H.O. behavior of the silica particles showed that strain 97AE3-12 6.4 g/L, and sodium citrate, 1 g/L. Separate media had as the formed a strong adhesive interaction with adjacent crystalline electron acceptor either NaNO. 2 g/L or NaFumarate 3.7 silica particles causing clumping of the particles. Aggregation g/mL. The Salinity is 64 ppt. occurred for strain 97AE3-12 cultures in both nitrate and 0191 Duplicate samples of each medium were inoculated fumarate media, though more aggregation occurred with with 200 uL of an aerobic culture of strain 97AE3-12. Dupli nitrate as the electron acceptor.

TABLE 10 Settling of silica particles due to microbial induced particle aggregation. Optical density Optical density Optical density Optical density (OD), 600 nm, (OD), 600 nm, Electron Donor (OD), 600 nm (OD), 600 nm, 1 minute 10 minutes and Acceptor Treatment before mixing after mixing after mixing 1000 ppm uninoculated control, #1 -O.OO13 O4597 Citrate uninoculated control, #2 O.OO13 O.4794 2000 ppm Mean O O4696 US 2012/0277127 A1 Nov. 1, 2012

TABLE 10-continued Settling of silica particles due to microbial induced particle aggregation. Optical density Optical density Optical density Optical density (OD), 600 nm, (OD), 600 nm, Electron Donor (OD), 600 nm (OD), 600 nm, 1 minute 10 minutes and Acceptor Treatment before mixing after mixing after mixing NaNO, inoculated test #1 O.O852 O.1738 0.1527 inoculated test #2 O.O826 O.1393 O112 Mean O.O839 O.1566 O-1324 % reduction in OD Not applicable 66.7% 71.0% 1000 ppm uninoculated control, #1 O.O11 0.444 O.339 Citrate uninoculated control, #2 O.O11 O.486 O.360 3500 ppm Mean O.O11 O.465 O3SO NaFumarate inoculated test #1 O.018 O.26S O.217 inoculated test #2 O.O2S O.272 O.213 Mean O.O21 O.268 O.215 % reduction in OD Not applicable 42.3% 38.6%

Example 8 about 25 days (ending on day 45, FIG. 8). The pressure drop Control Slim Tube Pressure Drop Measurements was measured across slim tube 9a throughout the regiment of pulse feeding. Four hr pulses were fed on days, 20, and 24. (0193 The slim tube set-up described in General Methods Eight hr pulses were fed on days 27, 32, 34, 41, and 44. was used to measure pressure changes of a control sand sample over time. Brine #1 that had been filter sterilized was Between each of these nutrient pulses Brine #1 was fed at a fed continuously 8 days to slim tube 9a while the pressure rate of 3.6 ml/hr. The pressure drop was initially about 3 psi drop across the slim tube was measured (day 4 through day 12 (0.0207 mega Pascal). Ten days after initiating pulse feeding in FIG. 7). The pressure drop remained about 3 psi (0.0207 on slim tube 9a there was a discernable increase in pressure mega Pascal). This illustrates the stability of the packed sand drop that became more pronounced with time (FIG. 8). At the in the slim tube while being flooded with the filtered injection end of the experiment on day 45 the pressure drop was nearly brine, as no change in the pressure drop across the slim tube 3 times the control (Example 8). At this point in time an was observed experimentally. This is contrast to the treated effluent sample was taken and cell counts were measure and slim tubes described below in Examples 9 and 10 that showed are shown in Table 11 (Cell Count 4: 9a). This substantial marked changes in pressure drop as a result of the microbial increase in pressure demonstrates the potential for Arco treatment. bacter sp. 97AE3-12 (ATCC NO: PTA-11409) to effectively Example 9 modify the permeability of porous rock when it is fed batch wise with nutrients. Inoculated, Batch Fed Slim Tube Pressure Drop Measurements Example 10 0194 Slim tube 9a of Example 8 was pre-inoculated with 60 ml of live injection water (Brine #1 which was not filter Continuously-Fed Core Sand Slim Tube and Pres sterilized) at a rate of 15 ml/hour for 4 hours. Following this sure Drop Measurements pre-inoculation, an effluent sample was collected from slim tube 9a and cell counts were measured and are shown in Table (0197) Slim tube 9b (General Methods) was pre-inoculated 11 (Cell Count 1: 9a). 0.195 One day after pre-inoculation with unfiltered live with live injection water from the test well site (unfiltered injection water (Brine #1), the slim tube was inoculated with Brine #1), sampled for cell counts in the effluent of the slim strain 97AE3-12 (ATCC NO: PTA-11409). A 1:1 dilution of tube (Table 11, Cell count 1:9b), and inoculated with strain a growing culture of strain 97AE3-12 with unfiltered live 97AE3-12 as in Example 9, except that Arcobacter sp. strain injection water was grown in Brine #5 and incubated at room 97AE3-12 was not diluted 1:1 with unfiltered injection Brine temperature for 48 hours with agitation, and then diluted 1:30 #1. The cell count was measured in this inoculum and is in Brine #5 to inoculate slim tube 9a. Cell counts of this shown in Table 11 (Cell Count 2:9b). Following inoculation inoculum were determined and are shown in Table 11 (Cell with Arcobacter sp. strain 97AE3-12 the slim tube 9b was Count 2: 9a). A 50 ml volume of this inoculum was pumped aged for 6 days as above. An effluent sample was taken and into the slim tube at a rate of about 0.25 ml/min. The process cell counts were measured after inoculation and are shown in of slim tube inoculation took about 4h to complete. Follow ing inoculation the slim tube was shut in for 6 days. An Table 11 (Cell Count 3:9b). Brine #2 nutrient feed was effluent sample was taken after the 6 day shut in period and continuously fed to slim tube9b at a rate of 3.6 ml/hour for the cell counts were measured and are shown in Table 11 (Cell duration of the experiment while the pressure drop across it Count 3:9a). was measured (FIG. 9). Initially the pressure drop was about 0196. Starting on day 20, upon completion of the aging 2 psi (0.0137 mega Pascal) as shown in FIG. 9 (see day 20). period, Brine #2 was fed to slim tube 9a in 4 to 8 hr pulses at By day 32, the observed pressure drop for slim tube 9b had a rate of 3.6 ml/hour twice a week (once every 3 or 4 days) for increased by about a factor of 2 to 3 compared to the initial US 2012/0277127 A1 Nov. 1, 2012 22 pressure drop at day 20. At day 32.8, feeding of Brine #2 was stopped due to a pump failure. In order to restart the pump, it TABLE 11 had to be primed at a high flow rate. This pump priming operation appeared to reduce the pressure drop as seen after Cell counts in different slin tube experiment samples day 32.8 in FIG.9. However, after day 33, nutrient feed Brine Analysis Slim tube 9a Slim tube 9b #3 was fed again at a rate of 3.6 ml/hour and the pressure drop Cell count 1: effluent of slim tube 2.3 x 10 CFU/ml 2.3 x 10 CFU/ml climbed again so that it was about a factor of 2 higher at the following pre-inoculation with live end of the experiment at day 45 as compared to the initial brine pressure drop at day 20. At this point in time an effluent Cell count 2 of the Arcobacter 1.1 x 10 CFU/ml 1.7 x 10 CFU/ml sample was taken and cell counts were measure and are inoculum Cell count 3 in slim tube effluent 5.2 x 10 CFU/ml 7.6 x 10 CFU/ml shown in Table 11 (Cell Count 4: 9b). This substantial after 6 day aging with Arcobacter increase in pressure demonstrates the potential for Arco inoculum bacter sp. 97 AE3-12 (ATCCNO: PTA-11409) to effectively Cell count 4: MPNS in effluent 1.2 x 107 CFU/ml 3.1 x 10 CFU/ml modify the permeability of porous rock when fed continu ously with nutrients.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 52

<21 Os SEQ ID NO 1 &211s LENGTH: 1477 &212s. TYPE: DNA <213> ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: Arcobacter species <4 OOs SEQUENCE: 1 agagtttgat tatggct cag agtgaacgct gg.cggcgtgc ttaa.ca catg caagttcgaac 60 gagaacggga ttagcttgct aatctgtcag ctaagtggcg cacgggtgag taatatatag 12O

gtaacgtgcc ttcaa.gagggggata acaga tiggaaacgt.c tictaaaac C ccatatgc ct 18O ttaatgcgala agtatgcaag ggaaatattt at agcttgaa gatcggcctg tacagtatica 24 O

gatagttggit gaggtaatag ct caccalagt caatgacgct taactggttt gagaggatga 3 OO tdagt cacac tdgaactgag acacggit coa gact Cotacg ggaggcagda gtggggaata 360 ttgcacaatg ggggaalacc C tatgcagca acgc.cgc.gtg gaggatgaca catttcggtg 42O

cgtaaactic c ttittatataa galagataatg acgg tatt at atgaataagc accqigotaac 48O

to cqtgc.cag cago.cgcggit aatacggagg gtgcaa.gcgt tact cqgaat cactgggcgt. 54 O aaagagcgt.g. taggcggata gataagt cag aagtgaaat C caat agctta act attgaac 6 OO tgcttittgaa actgtctatic tagagtatgg gagaggtaga tiggaatttct ggtgtagggg 660 taaaatc.cgt agagat Caga aggaataccg attgcgaagg catct actg. galacatalact 72O gacgctgaga cqcgaaag.cg toggggagcaa acaggattag at accctggit agtic cacgc.c 78O

ctaaacgatg tacac tagtt gttgctatogc ticga catago agtaatgcag ttaa.ca catt 84 O

aagtgtaccg cctggggagt acggit cqcaa gattaaaact caaaggaata gacggggacc 9 OO cgcacaa.gcg gtggagcatg tdgtttaatt cacgatacg cgaagaacct tacctggit ct 96.O

tgacatagta agaac cattt agagatagat gggtgtctgc ttgcagaaac titat at acag 102O

gtgctgcacg gctgtcgt.ca gCtcgtgtcg tagatgttg ggittaagttcc cqcaacgagc 108O

gcaac cct cq to attagttg ctaac acttic gggtgagaac totaatgaga citgcctacgc 114 O

aagtaggagg aaggtgagga cacgtcaag to at catggc ccttacgacc agggct acac 12 OO

acgtgctaca atggggtata caaagagcag catacagtg atgtggagca aatctaaaaa 1260

US 2012/0277127 A1 Nov. 1, 2012 53

- Continued gatagttggit gaggtaatag ct caccalagt caatgacgct taactggttt gagaggatga 3OO t cagt cacac tdaactgag acacggtcca gactic ctacg ggaggcagca gtggggaata 360 ttgcacaatgggggaaac cc tatgcagca acgcc.gc.gtg gaggatgaca cattt cqgtg 42O cgtaaact cottittatataa gaagataatg acggt attat atgaataagc accqqctaac 48O tcc.gtgc.cag cagcc.gcggit aatacggagg gtgcaa.gcgt tact.cggaat cactgggcgt. 54 O aaagagcgtg taggcggata gataagticag aagtgaaatc caatagctta act attgaac 6OO tgcttittgaa actgtctatc tagagtatgg gagaggtaga tiggaatttct ggtgtagggg 660 taaaatcc.gt agagat Caga aggaataccg attgc galagg Catctact.g. galacatalact 72 O gacgctgaga cqcgaaag.cg tdgggagcaa acaggattag at accctggit agt cc acgc.c 78O ctaaacgatg tacact agitt gttgctatgc ticgacatago agtaatgcag tta acacatt 84 O aagtgt accg cctggggagt acggit cqcaa gattaaaact caaaggaata gacggggacc 9 OO cgcaca agcg gtggagcatg tdgtttaatt cacgatacg caagaacct tacctggtct 96.O tgacat agta agaaccattt agagatagat gggtgtctgc titgcagaaac titatatacag O2O gtgctgcacg gctgtcgt.ca gct cqtgtcg tagatgttg ggittaagttcc cqCaacgagc O8O gcaa.ccct cq t cattagttg ctaac acttic gggtgagaac totaatgaga citgcc tacgc 14 O aagtaggagg aaggtgagga cacgtcaag ticatcatggc cct tacgacc agggctacac 2OO acgtgctaca atggggtata caaagagcag catacagtg atgtggagca aatctaaaaa 26 O atacct coca gttcggattg tagt ctdcaa citcgactaca tdaagttgga atcgctagta 32O atcgtagatc agcaatgcta C9gtgaatac gttc.ccgggit Cttgtactica cc.gc.ccgt.ca 38O

Caccatggga gttgattt ca citcgaagcgg ggatgctaag at agctaccC. tccacagtgg 44 O aattagcgac tdggtgaag ticgtaacaag gta accg 477

<210s, SEQ ID NO 4 O &211s LENGTH: 1481 &212s. TYPE: DNA <213s ORGANISM: Unknown 22 Os. FEATURE: <223> OTHER INFORMATION: Arcobacter species 22 Os. FEATURE: <221 > NAMEAKEY: variation <222s. LOCATION: (72) . . (72) <223> OTHER INFORMATION: Base A72 exists in >1 reference sequence; may be absent in others 22 Os. FEATURE: <221 > NAMEAKEY: variation <222s. LOCATION: (83) . . (83) <223> OTHER INFORMATION: Base T83 exists in >1 reference sequence; may be absent in others 22 Os. FEATURE: <221 > NAMEAKEY: variation <222s. LOCATION: (208) ... (208) <223> OTHER INFORMATION: Base C2O8 was found in only 1 reference sequence and was absent in others 22 Os. FEATURE: <221 > NAMEAKEY: variation <222s. LOCATION: (1318) . . (1318 <223> OTHER INFORMATION: Base G1318 was found in only 1 reference sequences and was absent in others

<4 OOs, SEQUENCE: 4 O agtcgagcggymtggcticag agtgaacgct ggcggcgtgc tita acacatg Caagttctgaac 6 O gagaacggrW tatagottgc tat atctgtc. agctaagtgg CdCacgggtgagtaatrtat 12 O

US 2012/0277127 A1 Nov. 1, 2012 57

- Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer

<4 OOs, SEQUENCE: 46 gtaaaacgac ggc.cagt 17

<210s, SEQ ID NO 47 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Shewanella oneidensis

<4 OOs, SEQUENCE: 47 gCatacgc.cc tacgggggala agagggggac titt C 34

<210s, SEQ ID NO 48 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Shewanella 16S rDNA degenerate signature sequence with variable positions in region 2 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (22) ... (24) <223> OTHER INFORMATION: n=a or g 22 Os. FEATURE: <221 > NAME/KEY: misc feature <222s. LOCATION: (29).. (29) 223 OTHER INFORMATION: n=a or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: n=a, , c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (30) <223> OTHER INFORMATION: n=a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (31) ... (32) 223 OTHER INFORMATION: n=t or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (34) . . (34) 223 OTHER INFORMATION: n=a or c

<4 OOs, SEQUENCE: 48 gCatacgc.cc tacgggggala anningggginn nintn 34

<210s, SEQ ID NO 49 &211s LENGTH: 41 &212s. TYPE: DNA <213> ORGANISM: Shewanella oneidensis

<4 OOs, SEQUENCE: 49 tcggagtttg gtgtcttgaa cactgggctic tica agctaac g 41

<210s, SEQ ID NO 50 &211s LENGTH: 41 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Shewanella 16S rDNA degenerate signature sequence with variable positions in region 5 22 Os. FEATURE: <221 > NAMEAKEY: misc feature US 2012/0277127 A1 Nov. 1, 2012 58

- Continued <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: n=a or g 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (13) . . (13) <223> OTHER INFORMATION: n=a , c or g 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (14) . . (14) <223> OTHER INFORMATION: n=a c or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (18) ... (19) <223> OTHER INFORMATION: n=a or g 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (27) . . (27) <223> OTHER INFORMATION: n=g or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (28) ... (28) 223 OTHER INFORMATION: n=t or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (30) ... (31) 223 OTHER INFORMATION: n=t or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (32) ... (32) 223 OTHER INFORMATION: n=a or c

<4 OOs, SEQUENCE: 50 tcqgantttg gtnincttinna cactggnntn innaagctaac g 41

<210s, SEQ ID NO 51 &211s LENGTH: 96 &212s. TYPE: DNA <213> ORGANISM: Shewanella oneidensis

<4 OOs, SEQUENCE: 51 acaatggcga gtacagaggg ttgcaaagcc gcgaggtgga gctaatct ca caaagct cqt 6 O cgtagt ccgg attggagt ct gcaacticgac to catg 96

<210s, SEQ ID NO 52 &211s LENGTH: 96 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Shewanella 16S rDNA degenerate signature sequence with variable positions in region 8 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: n=c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: n=a, c, or g 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (10) ... (11) <223> OTHER INFORMATION: n=a, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (25) ... (25) <223> OTHER INFORMATION: n=a or g 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (33) . . (33) <223> OTHER INFORMATION: n=a or g 22 Os. FEATURE: US 2012/0277127 A1 Nov. 1, 2012 59

- Continued <221 > NAMEAKEY: misc feature <222s. LOCATION: (38) ... (38) <223> OTHER INFORMATION: n=g or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (39) . . (39) <223> OTHER INFORMATION: n=a, g, or t 22 Os. FEATURE <221 > NAMEAKEY: misc feature <222s. LOCATION: (48) ... (48) 223 OTHER INFORMATION: n=t or c 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (51) . . (51) <223> OTHER INFORMATION: n=c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (56) . . (56) <223> OTHER INFORMATION: n=c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (57) . . (57) <223> OTHER INFORMATION: n=a, c, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (58) ... (58) <223> OTHER INFORMATION: n=c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (73) . . (73) 223 OTHER INFORMATION: n=t or c 22 Os. FEATURE: <221 > NAME/KEY: misc feature <222s. LOCATION: (94) . . (94) <223> OTHER INFORMATION: n=a or g <4 OOs, SEQUENCE: 52 acaatgginnin intacagaggg ttgcnaagcc gcnaggtnina gctaatcnca naaaginningt 6 O cgtagt ccgg atnggagt ct gcaacticgac tocntg 96

What is claimed is: 5. The method of claim 1 wherein the electron acceptor is 1. A method for enhancing oil recovery from an oil reser at least one ionic salt of nitrite or a combination of at least one Vo1r compr1S1ng: salt of nitrite and at least one salt of nitrate. a) providing a composition comprising: 6. The method of claim 1 wherein the oil reservoir com prises at least one fluid having a concentration of salt of at i) at least one strain of Arcobacter belonging to Arco least about 30 parts per thousand. bacter clade 1; and 7. The method of claim 1 or 5 wherein the strain of Arco ii) a minimal growth medium comprising at least one bacter belonging to Arcobacter clade 1 comprises a partial electron acceptor; 16S rDNA sequence selected from the group consisting of b) providing an oil reservoir, SEQID NOs; 1,33, 34,35, 36, 37, and 38. c) inoculating the oil reservoir with the composition of (a) 8. The method of claim 7 wherein the strain of Arcobacter Such that the Arcobacter containing composition popu belonging to Arcobacter clade 1 is selected from the group lates and grows in the oil reservoir; and consisting of 97 AE3-3 (ATCC No. PTA-11410) and 97 AE3 d) recovering oil from the oil reservoir; 12 (ATCC No. PTA-11409). 9. The method of claim 1 wherein the composition of (a) wherein growth of the Arcobacter in the oil reservoir further comprises one or more additional microorganisms enhances oil recovery. which grow in the presence of oil under denitrifying condi 2. The method of claim 1 wherein the at least one strain of tions. Arcobacter belonging to Arcobacter clade 1 comprises the 10. The method of claim 9, wherein said one or more 16S rRNA degenerate consensus sequence of SEQID NO:40. additional microorganisms comprises a Shewanella species 3. The method of claim 1 wherein the at least one strain of or Thauera sp. AL9:8 (ATCC #PTA-9497). Arcobacter belonging to Arcobacter clade 1 comprises a 16S 11. The method of claim 10 wherein the Shewanella spe rDNA sequence having at least about 97% sequence identity cies comprises a 16S rDNA comprising the degenerate sig to a sequence selected from the group consisting of SEQID nature sequences of SEQID NOs: 48, 50 and 52. NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 39. 12. An isolated microorganism of a strain selected from the 4. The method of claim 1 wherein the electron acceptor is group consisting of 97AE3-3 (ATCC No. PTA-11410) and at least one ionic salt of nitrate. 97AE3-12 (ATCC No. PTA-11409). US 2012/0277127 A1 Nov. 1, 2012

13. An oil recovery enhancing composition comprising: 16. The composition of claim 13, further comprising one or a) at least one isolated strain of Arcobacter comprising a more additional microorganisms. partial 16S rDNA sequence selected from the group 17. The composition of claim 16, wherein said one or more consisting of SEQID NOs; 1,33, 34,35,36, 37, and 38: additional microorganisms grows in the presence of oil under b) one or more electron acceptors; and denitrifying conditions. c) at least one carbon Source. 18. The composition of claim 17, wherein said one or more 14. The composition of claim 13 wherein the strain of additional microorganisms comprises a Shewanella species Arcobacter is selected from the group consisting of 97AE3-3 or Thauera sp. AL9:8 (ATCC #PTA-949). (ATCC No. PTA-11410) and 97 AE3-12 (ATCC No. PTA 19. The composition of claim 18 wherein the Shewanella 11409). species comprises a 16S rDNA comprising the degenerate 15. The composition of claim 13 wherein said at least one signature sequences of SEQID NOs: 48, 50 and 52. carbon source is selected from the group consisting of lactate, acetate, formate and Succinate. c c c c c