The present invention generally concerns the field of oil engineering and more particularly the treatment of oil reservoir samples including outcrop samples during storage and/or before conducting tests on said samples.

Enhanced Oil Recovery (EOR) generally includes the injection of chemicals to assist the water flooding action of water. Injected chemicals include surfactants, polymers, various additives such as alkali, buffers, stabilizing agents, solubilizing agents, co-surfactants, clarifying agents, bactericides, scale inhibitors, anti-hydrate agents, etc...

EOR chemicals and conditions are determined on a case by case basis depending on each reservoir. To optimize the EOR chemicals and conditions to be implemented, reservoir samples are collected in the form of cores or outcrops which are in turn tested for physico-parameters such as wettability, adsorption of polymers, adsorption of surfactants, cation exchange capacity (CEC), etc...

When conducting such tests it is thus paramount to work on samples that are as representative as possible of the subterranean reservoirs conditions.

Reservoirs are generally reduced environments so that metals present in the minerals of the reservoirs are predominantly in their reduced form(s). Following sampling of reservoir core material, metals oxidize when contacted with oxygen. A similar problem exists for outcrop core material, which is often used in laboratory screening experiments due to the dearth of homogeneous reservoir core material. This material has typically been exposed to oxygen and is thus thoroughly oxidized.

This oxidation can have an important effect on surface physical chemistry of the sample, biasing measures of wettability (and thus likely relative permeability), CEC, surfactant and polymer adsorption.

Wang et al., SPE Reservoir Engineering, 1993, 108-1 16 reported that surfactant adsorption in the field may be up to ten times less than expected following laboratory assays on core samples, and that the oxidizing storage conditions of the core samples may explain the discrepancy. Wang and Guidry, SPE-Formation Evaluation, 1994, 140-148 further documented the role of oxidation-reduction potential in governing core wettability. In a general sense, the oxidation of iron (II) in the clay matrix, in minerals, or adsorbed on one of the two, yields a more positive surface, increasing affinity for anionic species such as the most common EOR surfactants and polymers, as well as acidic components of crude oils.

A difficulty in laboratory experiments consists in reproducing the reducing conditions present in oil reservoirs in the presence of ubiquitous oxygen. Another difficulty is to restore the oxidized core or outcrop sample in its reduced state as originally present in the reservoir.

To date, the method for sample preservation and/or reduction following coring is contacting the sample with sodium dithionite, a strong and highly unstable reducing agent. Drawbacks of this method include the dissolution of calcite and destruction of clays due to the evolution of acidity during the decomposition of sodium dithionite. The acidity may also substantially affect the measured parameters. Additionally, sodium dithionite has been demonstrated to be highly degrading to EOR polymers, leading to additional complications if subsequent polymer injection is a goal. Thus there remains a need for methods of obtaining reduced reservoir samples where the reduction method is environmentally non-hazardous and does not affect the measured parameters.

There is also a need for providing alternative, more practical methods for treating oil reservoir samples so as to preserve and/or restore their state as in the reducing conditions of the reservoirs.

One difficulty is that reservoir samples may be already oxidized to an unknown extent and may also contain oxygen or not. The process must be therefore highly versatile in that it must be able to optionally consume oxygen, if present, on the one hand and reduce any already oxidized metals, on the other hand.

US201 1 /003956 discloses the use of Shewanella to alter the interface of hydrocarbons and hydrocarbon-coated surfaces to increase oil recovery. In particular, Shewanella is disclosed as being allegedly able to affect the wettability of the reservoirs surface. However, US201 1 /003956 is only concerned with the improvement of oil recovery from reservoirs and neither addresses the issues of treating, preserving or restoring samples, nor the achievement of a reducing effect. Many microorganisms are capable of reducing metal oxyhydroxides. However, none of them have been ever considered for reducing oil reservoir samples.

The inventors have shown that oxidation of iron strongly affects the surface properties in reservoir cores and outcrops, in particular due to the formation of HFO (hydrous ferric oxide). In the reduced environment of the reservoir, iron predominantly exists in the form of its Fe ²⁺ cation, whereas in oxidized samples iron oxidizes into its Fe ³⁺ form.

It is thus desired to provide for a sample treatment that targets the selective and efficient reduction of iron.

The present inventors have identified the genus Shewanella, and more particularly the Shewanella putrefaciens species, notably an appropriate strain, herein called CN32 as well as the operating conditions which provide for an easy, reliable, effective treatment and storage of oil reservoir samples and allow both maintenance of a reducing environment for samples and restoration of a reduced state, if required. In particular, the CN32 strain has been shown to be a highly specific and efficient iron reducer.

The invention thus relates to a method for treating oil reservoir samples with said specialized iron reducing microorganisms belonging to the Shewanella genus. The invention also describes a method of culturing the specialized strain to outcompete other environmental microbes present in the reservoir to perform the desired iron reducing. In addition, iron ions can be coerced into secondary mineral formation of non-reactive iron containing minerals, which can be influenced by varying bicarbonate concentrations. According to an object, the present invention concerns a process for treating an oil reservoir sample comprising:

incubating one or more bacteria of the strain Shewanella CN32 (Shewanella putrefaciens LH4:18 (ATCC No. PTA 8822)) or a genetically modified mutant thereof with said oil reservoir sample, in the presence of an electron donor. According to a further object, the present invention also concerns a process for maintaining oil reservoir samples under reducing conditions comprising the process of treatment of the invention. According to a still further object, the present invention also concerns a process for restoring reservoir samples into reduced conditions comprising the process of treatment of the invention.

The process of the invention allows preserving reduced forms and environment and/or reducing atmospherically-oxidized minerals such as ferric oxides (iron oxyhydroxide HFO) present in reservoir samples.

The process of the invention may thus comprise the formation of reduced minerals more representative of those found in the reservoir, such as magnetite, pyrite, and siderite, rather than just elude Fe(lll) as soluble Fe(ll).

Advantageously, the process of the invention allows efficient reduction of iron oxyhydroxides. A portion of that iron has been shown to be in the oxidized Fe(lll) within the reservoir. Minerals (Fe ₂ 0 ₃ , Fe(OH) ₃ , magnetite and clays) can remain oxidized after burial into the crust of reservoir. Further, drilling often exposes the samples to oxygenated water or air. Further, it is believed that heavy oil is associated with bacterial activity which can oxidize Fe(ll) into Fe(lll). Also, surfactant adsorption has been strongly correlated with reactive iron oxyhydroxides. The expression "reservoir samples" used herein refer to samples that are intended to be representative of reservoirs. Typically, they are samples that are collected for obtaining information on the reservoir, from which or close to which they have been sampled. Reservoir samples include core samples and outcrops samples. According to an embodiment, the reservoir samples comprise Fe, Mn, Cr, In, U, and/or mixtures thereof, in particular Fe.

The process thus comprises the formation of an incubation medium which comprises the bacteria and the electron donor. It may comprise further ingredients as will be apparent below. Shewanella CN32 or CN32 as used herein refers to Shewanella putrefaciens LH4:18 (ATCC No. PTA-8822) strain of gram negative gamma proteo-bacteria species or genetically modified mutants thereof.

Genetically modified mutants include CN32 derivatives whose genome has been modified to be made resistant to antibiotics or antifungal agents, such as kanamycin, and/or natamycin.

The preparation of such mutants is well-known in the art. In particular they can be prepared by insertion in the bacterium plasmids comprising at least one antibiotic or antifungal resistance gene, such as AKN84 (PA1/04/03-ecfp cloned into Not! site of pBKminiTn7-KrnQSm1 ) as disclosed by Lambertsen et al (Environ. Microbiol. 6, 726-732, 2004). Description of general methods for cloning into plasmids and transforming bacteria reference is made in Molecular Cloning: A Laboratory Manual (Fourth Edition) Michael Green and Joseph Sambrook, 2012 Cold Spring Harbor Laboratory. The genus Shewanella is particularly well suited for metal reducing applications as it is a facultative anaerobe, which means that it can grow under both aerobic and anaerobic conditions.

The CN32 strain can reduce many metals, including Fe(lll), Co(lll), U(VI), Cr(VI), Tc(VII), and the Fe(lll); reduction kinetics have been previously characterised.

The inventors have now shown that CN32 may reduce iron with a more significant rate than the other Shewanella species or strains. Further, they have also shown that CN32 are specific for iron, as they congregate on iron specifically. CN32 can be incubated at temperature comprised between 20 and 40° C; in particular between 25 and 35°C. It is capable of growth in a wide variety of saline conditions from 1 to 30 % and will be initially grown in low salinity conditions of from 0.5 to 10%, such as around 1 .4%. As CN32 are facultative anaerobes, the process may be carried out under aerobic or anaerobic conditions.

Under aerobic conditions, oxygen functions as an electron acceptor and is hereby reduced. In the presence of oxygen, these facultative anaerobes would thus consume the oxygen first. Once oxygen is eliminated, these bacteria would reduce oxidized metals present in the sample. Under anaerobic conditions, oxidized metals present in the sample function as electron acceptors are hereby reduced.

It is to be understood that aerobic conditions refer to the presence or addition of oxygen, whereas anaerobic conditions refer to conditions where oxygen is not present.

When present in the sample, oxygen is thus the initial electron acceptor, which is consumed first. Oxygen carried in the sample may be present in pores of the sample; it may also be present in the incubation medium, or may also come from air.

When present, the oxidized metal(s) function as secondary electron acceptor.

According to an embodiment, the process may be thus conducted under aerobic conditions followed by anaerobic conditions.

According to an embodiment, oxidized metals comprise Fe(lll).

According to an embodiment, samples may be in air-tight containers such as sleeves. In such air-tight containers, the process may be started under aerobic conditions if oxygen is present in the sample or in the incubation medium or initially added to the sample, and the process may then go on under anaerobic conditions once the oxygen is consumed. Alternatively, the process may start anaerobically if the sample, the incubation medium and the air-tight container do not comprise oxygen.

According to an embodiment, the process of the invention thus further comprises adding an initial additional electron acceptor to the incubation medium to initiate the incubation.

Said initial additional electron acceptor may be oxygen or an organic electron acceptor. Organic electron acceptors may be selected from the group consisting of trimethylamine N-oxide, dimethyl sulfoxide, nitrite, nitrate, fumarate and mixtures thereof. According to an embodiment, the process can be carried out on a non-oxidized sample, such as immediately following sampling or on a sample that has been anaerobically stored straight after sampling. The process will thus maintain the sample in a reduced form, representative of the original reservoir conditions. According to another embodiment, the process can be conducted on an at least partially oxidized sample, such as samples stored without special precautions for an extended time or outcrop samples.

Said oxidized samples may comprise oxidized iron minerals and/or colloids.

According to an embodiment, the process of the invention may comprise the reduction of iron (III) into iron (II), following the optional consumption of oxygen and/or the initial additional electron acceptor. Said oxygen and/or initial additional electron acceptor may thus also be called hereafter "transitional electron acceptor".

According to an embodiment, the process may comprise the reduction of an initial electron acceptor prior to reduction of iron (III). Said initial electron acceptor may be oxygen present in the sample or in the incubation medium or can be an initial additional electron acceptor as defined above.

According to an embodiment, the electron donor is a carbon source. It may be chosen so as to maximize the energy released (per mole) in oxidation reactions, and to comply with metal reduction reaction.

Electron donors may be selected from the group consisting of: lactate, formate, pyruvate, particularly lactate.

In the course of the process, the samples may be coated with a non-reactive biofilm formed by the bacteria and/or bacterial cells.

According to the invention, it was shown that the combination of the reduction and biofilm achieved a potentialized action in the reconditioning of oil reservoir sample.

According to an embodiment, the process may also comprise the step of removing biofilm. The biofilm does not prevent reduction of the sample and can be maintained. However, it is preferred to remove the biofilm prior to conducting experiments on the samples.

The process of the invention may be conducted according to batch or continuous flow design. The sample incubation may be continuously fed in chemostat culture and liquid waste products continuously removed (b) or may be pulse fed at the end of incubation period. According to an embodiment, the incubation medium comprises a broth suitable for growing bacteria.

There are many different broths for growing bacteria. A typical example of nutriment broth suitable for the culture of Gram negative bacteria is LB medium (Bertani, G. (1951 ). J. Bacteriol. 62:293-300).

In particular the incubation medium comprises a nutritive medium. Said nutritive medium will provide all nutriments needed by the bacteria for growth. According to an embodiment, said nutritive medium is a minimal medium in that it provides the minimal nutriments for growth.

According to an embodiment, the nutritive medium may contain additional components generally used to support microorganism growth, which may include vitamins, trace metals, nitrogen, phosphorus, magnesium, calcium, and/or buffering chemicals. According to an embodiment, the minimal medium comprises vitamins, aminoacids, minerals, buffer, alkali, salts. Further minimal media including amino acids, vitamins, minerals, and carbons source/electron donors may be selected to minimize variability and optimize growth conditions. The medium can be used in any form. In particular, it can be a ready-to-use pre-mixed dry medium.

It may be desired to sterilize the sample and/or the incubation medium. According to an embodiment, autoclaving (high pressure and temperature of 121 °C) for sterilization of samples will be avoided in order to prevent unintentional alteration of mineral composition and crystal structure.

The reservoir sample may be sterilized using isopropyl alcohol prior to introducing cell culture in the incubation medium.

Alternatively, in non-sterile samples, biological contaminants can be minimized by addition of known antibiotics, such as kanamycin, and/or antifungals such as natamycin, into the media. CN32 can generally grow in the presence of natamycin. Alternatively or additionally, CN32 can be made resistant to kanamycin by genetic transformation. In particular, the construct may be made by inserting plasmids for these agents, such as AKN84 {PA1 /04/03-ecfp cloned into Notl site of pBKminiTn7-KmQSm1 ) as disclosed by Lambertsen et al Environ. Microbiol. 6, 726-732, 2004. However, a large number of plasmids containing antibiotic or antifungal resistance gene are commercially available and could be used instead. General methods for transforming bacteria with plasmids are known in the art, and described for instance in Molecular Cloning: A Laboratory Manual {Fourth Edition) Michael Green and Joseph Sambrook, 2012 Cold Spring Harbor Laboratory. The process of the invention allows for secondary mineral formation (biomineralisation). Some of these secondary minerals commonly include siderite, pyrite, magnetite and hematite. Secondary mineral formation can be influenced by the pH, the buffer, the nature and concentration. The process of the invention may allow control of the secondary mineral formation in particular by adjusting the pH, such as with a buffer, e.g. a bicarbonate buffer, with a concentration that is between about 10 ppt and 55 ppt.

According to an embodiment, the concentration of the bacteria in the incubation medium may be comprised between 50 and 500 cells/mL. To achieve this concentration, cells may be grown to an ultra-high density and then concentrated by centrifugation.

Before use, the live bacteria can be used in any form, such a pellet, in particular dissolvable gel pellets.

According to an embodiment, the process of treatment of the invention can be carried out prior to conducting laboratory experiments on the reservoir samples, such as wettability measurement, relative permeability measurement, CEC measurement, chemical adsorption measurement, or other core flood or EOR/IOR experiment.

According to a further object, the present invention also concerns a process for storing an oil reservoir sample comprising:

a. carrying out the process for treating said sample according to the invention; and

b. maintaining the sample under anaerobic conditions.

According to a further object, the present invention also concerns a method for assaying an oil reservoir sample comprising:

a. carrying out the process for treating said sample according to the invention;

b. removing the biofilm that may have formed on the sample; and c. conducting the assay. The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.

Applicants specifically incorporate the entire content of all cited references in this disclosure. Unless stated otherwise, all percentages, parts, ratios, etc., are by weight. Trademarks are shown in upper case. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.

The following definitions are provided for the special terms and abbreviations used in this application:

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion.

The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

"Petroleum" or "oil" is a naturally occurring, flammable liquid found in rock and sand formations in the Earth, which consisting of a complex mixture of hydrocarbons and polycyclic aromatic hydrocarbon of various molecular weights, plus other organic compounds.

The term "oil reservoir" refers to a subterranean or subsea-bed formation from which oil may be recovered. The formation is generally a body of rocks, consolidated sand and soil having sufficient porosity and permeability to store and transmit oil.

The term "well bore" refers to a channel from the surface to an oil-bearing stratum with enough size to allow for the pumping of fluids either from the surface into the oil-bearing stratum (injection well) or from the oil-bearing stratum to the surface (production well). The term "Microbial Enhanced Oil Recovery" (MEOR) is a biological based technology consisting in modifying microbial function or structure, or both, of microbial environments or microbes, or both existing in oil reservoirs. The ultimate aim of MEOR is to improve the recovery of oil entrapped in porous media. MEOR is a tertiary oil extraction technology allowing the partial recovery of residual of oil in effect, increasing the life of oil reservoirs. The term "electron donor" refers to a molecular compound that gives or donates an electron(s) during cellular respiration.

The term "electron acceptor" refers to a molecular compound that receives or accepts an electron(s) during cellular respiration. Microorganisms obtain energy to grow by transferring electrons from an "electron donor" to an "electron acceptor". During this process, the electron acceptor is reduced and the electron donor is oxidized. Examples of electron acceptors include oxygen, nitrate, fumarate, iron (III), manganese (IV), sulfate and carbon dioxide. Sugars, low molecular weight organic acids, carbohydrates, fatty acids, hydrogen and crude oil or its components such as petroleum hydrocarbons or polycyclic aromatic hydrocarbons are examples of compounds that can act as electron donors. The terms "denitrifying" and "denitrification" mean reducing nitrate or nitrite for use in respiratory energy generation.

"Adhered to" refers to coating or adsorption of a liquid to a solid surface of at least 10% areal coverage.

The term "wetting" refers to the ability of a liquid to maintain contact with a solid surface, resulting from intermolecular interactions when the two are brought together. The degree of wetting (expressed as "wettability") is determined by a force balance between adhesive and cohesive forces.

"Wetting agent" refers to a chemical such as a surfactant that increases the water wettability of a solid or porous surface by changing the hydrophobic surface into one that is more hydrophilic. Wetting agents help spread the wetting phase (e.g. water) onto the surface thereby making the surface more water wet.

"Interface" as used herein refers to the surface of contact between a water layer and an oil layer, a water layer and a solid surface layer, and an oil layer and a solid surface layer. "Hydrocarbon-coated" as used herein refers to a coating of a hydrocarbon to a solid surface of at least 10% areal coverage.

The term "components of a subsurface formation" refers to rock, soil, brine, sand, clay or mixtures thereof of either subterranean or seabed formations, that have come in contact with one or more hydrocarbon. These components may be part of an oil well or reservoir. At least a portion of the components include some hydrocarbon-coated surfaces, including particles with coated surfaces.

"Wettability" refers to the preference of a solid to contact one liquid, known as the wetting phase, rather than another. Solid surfaces can be water wet, oil wet or intermediate wet. "Water wettability" pertains to the adhesion of water to the surface of a solid. In water-wet conditions, a thin film of water coats the solid surface, a condition that is desirable for efficient oil transport.

The term "water flooding" refers to injecting water through well bores into an oil reservoir. Water flooding (secondary oil recovery) is performed to flush out oil from an oil reservoir when the oil no longer flows on its own out of the reservoir.

The term "biofilm" means a film or "biomass layer" of microorganisms. Biofilms are often embedded in extracellular polymers, which adhere to surfaces submerged in, or subjected to, aquatic environments. Biofilms consist of a matrix of a compact mass of microorganisms with structural heterogeneity, which may have genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.

The term "indigenous microorganisms" refers to the microorganisms that are native to the oil reservoir fluids and subterranean matrices.

The term "inoculated microorganisms" refers to the microorganism that are introduced to the oil reservoir fluids and subterranean matrices by injecting the microbes through a well bore into the oil reservoir substructure.

"Shewanella species" or "Shewanella spp." refers to microorganisms phylogenetically classified by rDNA typing to the Shewanella genus. Members to Shewanella are Gram negative, metal-reducing, gamma-proteobacteria that are capable of reducing a wide range of terminal electron acceptors. These microorganisms gain energy to support anaerobic growth by coupling the oxidation of H ₂ or organic matter to the redox transformation of a variety of multivalent metals, which leads to the precipitation, transformation, or dissolution of minerals.

The term "salt" includes any ionic compound that can create ions in water including, but not limited to KCI, SrCI, NaBr, NaCI, CaCI ₂ , and MgCI ₂ . Fumarate may be in the form of any fumaric acid salts, or fumaric acid itself may in this context be included in the term fumarate. Fumarate salts may include a monosodium or disodium salt, calcium salt, magnesium salt, ammonium or diammonium salt, potassium or dipotassium salt, hydrochloride salt, or hydrated forms of any fumarate acid salt. In one embodiment, the organic electron acceptor contains disodium fumarate (DSF).

Figures:

Figures 1 and 2 represent the location of bacterial cells on minerals with a combination of fluorescence and brightfield Apatome™ microscopy, on sand (Fig. 1 ) and iron oxyhydroxides (Fig. 2).

Figure 3 illustrates biofilm formation, iron reduction and adsorption over 10-day treatment with Wild type CN32 bacteria on Uganda core samples. Figure 4 illustrates iron reduction after 3 days incubation with bacteria, mutant or control abiotic Ugandan core sample.

Figures 5 and 6 illustrate biofilm measurement on various mineral samples. Figures 7 and 8 demonstrate the alteration of minerals in Ugandan core before and after CN32 bacteria treatment. SEM-XRD analysis of untreated Ugandan core sample highlights iron-containing minerals (Figure 7). After treatment with CN32 bacteria, XRD analysis indicates depletion in Ugandan core samples (Figure 8). Examples

GENERAL METHODS

The meaning of abbreviations are used in this application are as follows: "h" means hour(s), "min" means minute(s), "day" means day(s), "mL" means milliliters, "mg/mL" means milligram per milliliter, "L" means liters, "μΙ_" means microliters, "mM" means millimolar, "μΜ" means micromolar, "nM" means nanomolar, '^g/L" means microgram per liter, "pmol" means picomol(s), "° C." means degree s Centigrade, "F." means degrees Fahrenheit, "bp" means base pair, "bps" means base pairs, "mm" means millimeter, "ppm" means part per million, "ppt" means part per thousand, "g/L" means gram per liter, "mL/min" means milliliter per minute, "mL/h" means milliliter per hour, "cfu/mL" means colony forming units per milliliter, "g" means gram, "mg/L" means milligram per liter, "Kev" means kilo or thousands of electron volts, "psig" means per square inch per gram, "LB" means Luria broth, "rpm" means revolution per minute, "NIC" means non inoculated control. Growth of CN32 on the incubation medium

Sterilization and preparation method of oil reservoir mineral samples occurred prior to bacterial incubation. For example, Ugandan outcrop mineral (UOM) samples were collected from Total in Oilfield outcrop in Uganda. The samples were sieved using a 180 micron sieve to remove large particles and debris. The UOM sample is then sterilized in excess 70% isopropyl alcohol and then dried in 60° C until all isopropyl has evaporated. The untreated UOM sample was analyzed before bacteria treatment with XRD, Mossbauer spectroscopy and elemental analysis on SEM.

Adsorption measurements were made using a photo active surfactant, alkylbenzenesulfonate (P550), (Dow Chemicals, Ny, CN 23425). The sample was first dried for 2 hours in a 60 °C drying oven. Then sa mple was then weighed and divided into four 15ml_ Falcon tubes. Each tube contained ~ 500mg of sample but the exact weight is recorded. Four concentrations of surfactant are then added 250, 500, 1000 and 1500 ppm, at a volume of 6ml_. Each tube is subjected to vortex for 30 seconds and then spun in centrifuge (Heraeus Multifuge X3, Thermo Scientific, Waltham, MA, United States of America) at 5500 rpm for 20 min at 20° C. The samp le is then filtered with a 2 micron syringe filter (Millex-GP, Darmstadt, Germany) and measured with 240-300nm wavelength scan in Ultraviolet spectrophotometer (Uvikon, Serlabo Technologies, Entraigues sur la Sergue, France). The peak near 262 is recorded and the concentration of surfactant is calculated based on a standard curve. Care is taken with surfactant to avoid skin irritation and insure proper waste disposal.

Hach-Lange iron measurement (Kit method)

The procedure described by the Hach-Lange kit for measurement range of 0.005 to 2mg/L was followed (LCW 021 , Hach-Lange GMBH, Dusseldorf, Germany). The samples were first spun at 5,500 x g for 20 min at 20°C. Then i nside an anaerobic chamber, each 5mL sample was filtered using a 0.22 micron filter syringe and added to spectrophotometer cuvette. Cuvettes must be thoroughly cleaned with iron free distilled water and 30% HCI before using. 0.2mL of acid reagent A is mixed in with sample by pipetting and then allowed to sit for 3min. Then 0.3mL of buffer solution B is mixed in with the sample solution. Iron-trace microcap containing ferrozine™ is then added and mixed by inversion. After 15min, the absorbance of the sample at 560nm is measured in spectrophotometer. Special care is taken to avoid air bubbles in the bottom of the cuvette.

Ferrozine Measurement of ferrous and ferric iron (Harris method)

Colorimetric ferrozine (PDT disulfonate; 3-[2-Pyridyl]-5,6-dip enyl-1 ,2,4-triazine-4,4'- disulfonic acid) was dissolved in 50 mM HEPES buffer and balanced to pH 7 at a concentration of 0.05% wt/vol. The ferrozine solution is then used before one month to prevent degradation. The sample is centrifuged for 20min at 10,000 rpm to remove all Fe(IV) and then mixed with equal volumes of saturated HCI (1 M). A separate sample with solid Fe(IV) is mixed with equal parts saturated hydroxylamine/HCI solution. The acidified sample is then mixed by vortex for 10 seconds and incubated at 60° C for 2h. Then 300 μΙ_ of acidified sample is transferred into 700 μΙ_ of ferrozine solution. Absorbance was measured in spectrophotometer (Novaspec Plus, Amerisham Biosciences, Little Chalfont, United Kingdom) at 562 nm using a 1 mL semi-micro cuvette (Ratiolab, Dreieich, Germany). This method has been adapted from Stookey L.L. 1970.

Crystal violet biofilm assay

Solid is removed sample and air dried at 60° C for 1 h. A (700mg) fraction of the solid sample is then placed into one well of a 26 well (3ml_) plate. Crystal violet stain is added to each sample well (125 μΙ_) and mixture is vortexed for 10 seconds. The sample well is then washed 10 times with 1 ml_ of dDI water. Then each sample is incubated with 200 μΙ_ of acidic acid (1 M) and allowed to sit for 15min. The volume of 125 μΙ_ is then added to sterile Dl water to total 1000 μΙ_ then placed in spectrophotometer cuvette. Absorbance was measured with spectrophotometer at 560 nm wavelength.

Biofilm washing

Two types of washing procedures were used to remove biofilm; Dl-vortex wash, and Dl- acid-vortex wash. For the Dl vortex procedure, the mineral sample was prepared using the above adsorption protocol and once dry, 10 mL of Dl water was added to mineral and the solution vortexed for 60 seconds, then the liquid was disposed of after treatment with a 30% bleach solution. This cycle, including vortex, was repeated 2 additional times. For the Dl acid treatment the procedure was identical except the first rinse contained 10mM of acidic acid and the solution was allowed to sit with the sample for 15 min. Then the remaining two rinses contained only Dl H ₂ 0. Levels of biofilm formation on minerals was quantified using the crystal violet biofilm assay (ref). Solid is removed, the sample and air are dried at 60°C for 1 h. A (700mg) fraction of the solid sample is then placed into one well of a 26 well (3mL/well) plate. Crystal violet stain is added to each sample well (125 μΙ_) and mixture is vortexed for 10 seconds. The sample well is then washed 10 times with 1 ml_ of dDI water. Then each sample is incubated with 200 μΙ_ of acidic acid (1 M) and allowed to sit for 15min. The volume of 125 μΙ_ is then added to sterile Dl water to total 1000 μΙ_ then placed in spectrophotometer cuvette. Absorbance was measured with spectrophotometer at 560 nm wavelength. Cultivation and strains

Cultivation of CN32 strain for domination of reservoir microbial populations was achieved by super concentration of cells into inoculation pellets. Shewanella putrefaciens CN-32, were utilized in this method. All cultures and mineral outcrop incubation experiments used a previously described defined minimal medium (M1 ), containing 18 mM lactate as an energy source, Harris et al, 2012, Biochemical Society Transactions 40(6), 1 167-1 177. Strains were inoculated from freezer stock onto Luria-Bertani (LB) plates and then grown overnight at 30Ό. Individual colonies were then se lected and inoculated into defined minimal media and grown overnight (M1 ). The cells were then harvested at 0.5 O.D. and then spun at 5,500 rpm for 20 min at 20Ό. All str ains were then resuspended in 150 μί of minimal media and then added to insoluble outcrop mineral sample (15 g), and 20 mL of media in 50 mL Falcon tubes (VWR International LLC, Randor, Pennsylvania, USA) and incubated horizontally in a shaker (180 rpm) for 1 -7 days at 30° C (Amerex Instruments, Lafayette, California, USA). Anaerobic samples where incubated on revolving wheel at 10 rpm in anaerobic chamber (company).

SEM Preparation

Outcrop mineral samples were removed and immediately fixed aerobically using 2% glutaraldehyde solution. Mineral outcrop (sand) was separated from liquid media with sterile metal scapula and then gently flushed (1 mL added then removed) with pH 7 PBS. The samples are then flushed with 50% PBS, and then sterile deionized water. Samples were then inundated with 500 μί ethanol then allowed to dry for 30min (in 10%, 50%, and then 100% concentrations of ethanol). The samples are then placed in a vacuum chamber overnight. The dried samples were coated with gold and then viewed by using a Zeiss- LEO 982 FE-SEM. HFO preparation

The Fe(OH) ₃ stock solution was prepared according to the protocol by Cornell and Schwertmann 2007 73(21 ), 7003-12, and then verified by X-ray diffraction.

Sterilization and preparation of Mineral samples

Ugandan outcrop mineral (UOM) samples were collected from the Kisegi Formation outcrop in the Semliki area, Uganda. The samples were sieved using a 180 micron sieve to remove large particles and debris. The UOM sample is then sterilized in excess 70% isopropyl alcohol and then dried in 60° C until all isopropyl has evaporated. The untreated UOM sample was analyzed before bacteria treatment with XRD, Mossbauer spectroscopy and elemental analysis on SEM. The sample was determined to contain 3% total iron, including a large proportion in the oxidized mineral Jarosite, as well as pyrite and smectite, which may contain additional oxidized iron as a surface coating or, in the case of smectite, as structurally-incorporated iron(lll). Mossbauer analysis revealed that approximately 97% of iron in this sample was oxidized.

The media

The minimal media was prepared according to protocol in Harris et al, 2012, Biochemical Society Transactions 40(6), 1 167-1 177. Initially NaOH is added to ¼ the working volume of the solution and PIPES buffer is dissolved into solution. Lactate can be highly viscous and is therefore weighed in Teflon weight dish. Vitamins, minerals and amino acids cannot be autoclaved. Instead sterile filtrate (0.22microns) in sterile hood all the amino acids, minerals, and vitamins and add to media after cooling the autoclaved solution.

Supplier and Final concentration

Chemical description

catalogue number in medium (mM)

Pipes buffer Sigma P-1851 50

Sodium hydroxide Sigma S-5881 7.5

Ammonium chloride Sigma A-5666 28.04

Potassium chloride Sigma P-4504 1 .34

Sodium phosphate

monobasic, Sigma S-9638 4.35

monohydrate

Vitamin solution, 100χ See below stock

Amino acid solution,

See below

100x stock

Mineral solution, 1 00 ^χ

See below

stock

Sodium lactate, 60%

Sigma L-1375 18

(w/w) syrup

(b) Vitamin solution

Supplier and Final concentration

Chemical description

catalogue number in medium (nM)

Biotin (D-biotin) Sigma B-4639 81 .87

Folic acid Sigma F-7876 45.34

Pyridoxine HCI Sigma P-9755 486.38

Riboflavin Sigma R-4500 132.84

Thiamine HCI Sigma T-4625 140.73

Nicotinic acid Sigma N-4126 406.17

D-Pantothenic acid,

Sigma P-2250 209.82

hemicalcium salt

Vitamin B ₁₂ Sigma V-2876 0.74

p-Aminobenzoic acid Sigma A-9878 364.62

Thioctic acid (a-lipoic

Sigma T-5625 242.37

acid)

(c) Amino acid solution

Concentration of Supplier and Final concentration

Chemical description

100x stock (g/L) catalogue number in medium (mg/L)

L-Glutamic acid 2 Sigma G-1251 2

L-Arginine 2 Sigma A-3909 2

DL-Serine 2 Sigma S-4375 2

(d) Mineral solution

Supplier and Final concentration

Chemical description

catalogue number in medium (μΜ)

Nitrilotriacetic acid Sigma N-9877 78.49 (dissolve with NaOH to

PH 8)

Magnesium sulfate

Aldrich 23,039-1

heptahydrate

Manganese sulfate

Aldrich 22,128-7

monohydrate

Sodium chloride Sigma S-3014

Ferrous sulfate

Sigma F-8633

heptahydrate

Calcium chloride

Sigma C-3881

dihydrate

Cobalt chloride

Sigma C-3169

hexahydrate

Zinc chloride Sigma Z-3500

Cupric sulfate

Sigma C-6283

pentahydrate

Aluminium potassium

Sigma A-7167

disulfate dodecahydrate

Boric acid Sigma B-6768

Sodium molybdate

Aldrich 22,184-8

dihydrate

Nickel chloride

Sigma N-6136

hexahydrate

Sodium tungstate Sigma S-0765

Preparation of electrocompetent CN32 cells

Before the AKN84 plasmid can be transformed into cells, electrocompetent CN32 cells must be prepared. The protocol below can be performed at room temperature of 25° C. The CN32 cells were grown on 5ml_ of LB media at 30° C, 185 rpm, in 15 mL falcon tubes with 10 mL of headspace to an OD600 of 0.4. Cells were then centrifuged at 6000 rpm for 5 minutes and washed with HEPES buffer. Washing is defined as discarding the supernatant then gently resuspending the cell pellet with 5 mL of 1 mM HEPES (pH 7). Centrifuge cells at 6000 rpm for 5min and wash once with 20% glycerol. Further centrifuge cells at 6000 rpm for 5min, discard supernatant and then add 10% glycerol until the final suspension reaches OD600 of 1 .3. The suspension is then distributed into 1 .5ml_ freezer vials in 100μΙ_ aliquots and stored at -80° C until transformation.

Plasmid preparation and purification

Plasmid is amplified by growing E.coli cells with AKN84 plasmid overnight in 5mL LB at 30° C at 185 rpm. Plasmid DNA was purified from E.coli using a commercial DNA purification kit and recommended protocol (QIAprep Spin Plasmid kit, Qaigen, Chatsworth, CA, USA). In an effort to improve yield, DNA was eluted from column with 25 μΙ of warm eiution buffer.

Transformation of AKN84 p!asmids into CN32 cells

Protocol for transforming AKN84 plasmid into CN32 cells proceeded only after electrocompetent CN32 cells are produced and plasmid DNA was isolated. The 100 μΙ aliquots of electrocompetent cells are removed from -80 Ό storage and placed on ice and used within 25 min. The electro cuvettes are then pre-chilled in refrigerator at 4°C. 5 μg of plasmid DNA (50 ng/μΙ) was added along with 100μΙ_ of chilled electrocompetent cells into electroporation cuvette (0.2 cm electrode gap). The suspension was mixed by gentle re- pipetting 20 times. Cuvette was chilled on ice for 1 min and then placed immediately in a BioRad GenePulse unit and electroporated.at 1 .1 kV. After electroporation the entire mixture was immediately added to 250μΙ_ SOC medium on ice in a 2ml_ cryovial. Cells were allowed to recover in 30° C for 2h on a rotating platform at 150 rpm. Cell mixture was plated on LB agar with antibiotic selection marker (50 nM kanamycin). Plates were incubated at 25° C for 1 -3 days. Colonies were then re-inoculated on LB kan plates and made into frozen stock.

Preparation of incubation medium comprising of kanamycin and natamycin

Kanamycin (kan) media was used within 3 weeks of preparation. Filter sterilization of kan stock, rather than autoclave sterilization, was used to prevent degradation of antibiotic. After the M1 media is prepared and autoclaved, as described in above section, 25 nM of kan and natamycin is filtered and added to 25° C media. The media is then kept refrigerated (4° C).

Example 1 Cells attached preferentially to Iron oxyhydroxides minerals in Ugandan outcrop

By using a combination of fluorescence and brightfield Apatome™ microscopy, the location of bacterial cells on minerals was determined (Figures 1 and 2). Fig. 1 shows brightfield image overlayed with fluorescence image of DAPI stained cells in 3 dimensions. Sand particles appear to be clear on the left side of Fig. 1 while iron oxyhydroxides appear darker and red in colour (center). The Fig. 2 shows only fluorescent image of DAPI stained cells with the black scale bar on the right = 40μηι. This analysis revealed that cells attached to Fe(OH) ₃ in density of 1 .5 cells/micron ² . The cells did not appear attached to Si (sand) particles or other non-iron oxyhydroxides containing minerals in the sample. The iron-oxide containing minerals were characterized with a distinctive red/brown color, while other minerals appeared clear or dark black without color. Non metal reducing bacteria strains, Pseudomonas fluorescens and AcymA (deletion mutant) did not attach to the iron oxyhydroxide containing mineral in high numbers (< 0.05 cells/micron ² ), florescence.

While high numbers of CN32 cells were found to attach to surface of Fe(OH) ₃ and HFO after 7 days of incubation (Figure 2), the cells did not appear to attach directly to Si mineral surface, as in the case of incubations with Ottawa sand, and Sikasol sand samples.

Example 2 Biofilm increases through time during incubation

Over a 10-day incubation with Ugandan outcrop core sample it was found that both in CN32 and non-metal reducing deletion mutant (AcymA) showed measurable levels of biofilm increase (Figure 3). It was also verified that high absorbance measures from crystal violet biofilm assays strongly correlated with high number of cells attached to minerals (data not shown). Measurements of biofilm also are significant in incubations with wild type (WT) CN32 with HFO, Ottawa sand, and UO rich samples (discussed below).

Example 3 Iron reduction correlated with reduced surfactant adsorption during incubation As apparent from Figure 3 (Adsorption (♦) reduction is correlated with more strongly with iron reduction (X) than biofilm formation (·)), bacterial reduction of iron oxyhydroxides by

CN32 (Fe(OH) ₃ ) was found in Ugandan outcrop to increase significantly, from 0.05mg/L to almost 20mg/L after 10 days of incubation in anaerobic conditions. Significant iron reduction of 15mg/L is detectable after only 3 days of incubation with CN32 strain using Hach-Lange Ferrozine method. Low levels of iron reduction (< 8mg/L) still occur in deletion mutant AcymA after 7 days of incubation. No significant abiotic reduction of Ugandan core samples was detected during this 10-day incubation. Example 4 Deletion mutant bacteria show insignificant Iron reduction

Both AcymA incubation and abiotic control show minor iron reduction in Ugandan outcrop sample relative to CN32. Fe(ll) concentrations determined by Hach-Lange Ferrozine assay can be seen in Figure 4. CN32 reduces almost 45% of total iron reduced by chemical reduction of Ugandan outcrop sample by concentrated HCI.

Example 5 Bicarbonate buffer impacts secondary mineral formation and reduction rate Three bicarbonate buffer concentrations were tested to determine the effects of secondary biomineralization. The effects of these three concentrations of bicarbonate on the biofilm formation can be seen in Figure 6. SEM and Mossbauer analysis of samples confirm mineral change. From elemental analysis the mineral was identified as siderite: a mineral not present in sample prior to treatment.

Example 6 Biofilm flush protocol reduces biofilm while maintaining adsorption benefits Washing of bacterial treated samples with non-invasive deionized water (Dl) flush and vortex regimen are compared to more chemically invasive 10mM acid and Dl flush with vortex regimen. The Dl flush reduced the quantity of biofilm down the level of abiotic control, while Dl/acid protocol reduced biofilm measure only slightly more significantly. The adsorption of these flushed core samples stayed significantly reduced over abiotic untreated sample (Figures 7).

Example 7 Bacteria treated samples lack Iron oxyhydroxides

Samples treated with CN32 for > 3 days lack detectable Fe in XRD analysis. Untreated Ugandan outcrop mineral samples show significant trace of Fe (Figure 7). Through X-ray diffraction (XRD) analysis it was confirmed that bacteria treated samples lacked detectable Fe(ll) or Fe(lll) (Figure 8). Control abiotic samples, as well as the original sample, still contained detectable Iron (Figure 4 and 7). Because the Hach-Lange Ferrozine measurements detected Fe(ll) in the bacteria treated supernatant while the XRD did not detect Fe(lll) in the insoluble mineral phase, it can be concluded that the Iron was reduced by the bacteria.