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Applicability of Anaerobic Nitrate-Dependent Fe(II) Oxidation to Microbial Enhanced Oil Recovery (MEOR) Hongbo Zhu,† Han K. Carlson,‡ and John D. Coates*,†,‡ †

Energy Bioscience Institute, and ‡Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, United States S Supporting Information *

ABSTRACT: Microbial processes that produce solid-phase minerals could be judiciously applied to modify rock porosity with subsequent alteration and improvement of floodwater sweep in petroleum reservoirs. However, there has been little investigation of the application of this to enhanced oil recovery (EOR). Here, we investigate a unique approach of altering reservoir petrology through the biogenesis of authigenic rock minerals. This process is mediated by anaerobic chemolithotrophic nitrate-dependent Fe(II)-oxidizing microorganisms that precipitate iron minerals from the metabolism of soluble ferrous iron (Fe2+) coupled to the reduction of nitrate. This mineral biogenesis can result in pore restriction and reduced pore throat diameter. Advantageously and unlike biomass plugs, these biominerals are not susceptible to pressure or thermal degradation. Furthermore, they do not require continual substrate addition for maintenance. Our studies demonstrate that the biogenesis of insoluble iron minerals in packed-bed columns results in effective hydrology alteration and homogenization of heterogeneous flowpaths upon stimulated microbial Fe2+ biooxidation. We also demonstrate almost 100% improvement in oil recovery from hydrocarbon-saturated packed-bed columns as a result of this metabolism. These studies represent a novel departure from traditional microbial EOR approaches and indicate the potential for nitrate-dependent Fe2+ biooxidation to improve volumetric sweep efficiency and enhance both the quality and quantity of oil recovered.

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gies are limited by financial cost and loss because of adsorption, phase partitioning, and trapping.3−5 While similar technologies based on biological metabolites are available with potentially better economics, microbial EOR (MEOR) usually involves the use of biomass to plug high porosity, thief zones in heterogeneous rock matrices, diverting floodwaters to low permeability, poorly swept regions.2 However, biomass plugs are susceptible to physical breakdown because of thermal damage or pressure collapse, in addition to cell death, cell lysis, and biological predation. Furthermore, biomass plugs require continual substrate addition for stability and maintenance. Here, we investigate a unique approach of altering reservoir petrology to improve hydrology and volumetric sweep efficiency through the biogenesis of authigenic rock minerals. This process is mediated by anaerobic chemolithotrophic nitrate-dependent Fe(II)-oxidizing (NDFO) microorganisms, which precipitate iron minerals from the oxidation of soluble ferrous iron (Fe2+) to insoluble ferric oxides under anoxic conditions with nitrate as the sole electron acceptor.6 These stable biogenic minerals result in pore restriction and reduced pore throat diameter in rock matrix, reducing the permeability of the high permeable zones and homogenizing the aqueous flow distribution. In addition, because this technology is based

INTRODUCTION Although the use of non-traditional energy sources, such as bioethanol, solar, and wind, will increase substantially over the next 2 decades, predictions suggest that these will account for less than 10% of total energy by 2030 (U.S. Department of Energy; www.eia.doe.gov/oiaf/ieo/index.html). Thus, reliance on fossil energy will likely continue as the dominant source of transportation fuels in the near future. Aside from their environmental impact, much of the cost associated with crude oil recovery stems from the fact that current technologies recover only one-third of the original oil contained within reservoirs. In the United States alone, more than 300 billion barrels (47.6 Gm3) of oil remain unrecoverable because of economic limitations of conventional technologies.1 At current U.S. consumption rates (https://www.cia.gov/library/ publications/the-world-factbook/rankorder/2174rank.html), this represents almost 50 years of independent reserves. As such, new technologies to enhance entrapped oil recovery are needed to increase accessible oil reserves, providing robust independent energy resources. Current enhanced oil recovery (EOR) strategies are varied and include both thermal and non-thermal methods. Thermal approaches involve heating the formation to reduce oil viscosity and improve its mobility. Non-thermal methods are either chemical or microbial.2 Chemical technologies generally involve synthetic surfactants to aid in oil solubility and mobility or polymers that alter floodwater viscosity and enhance injected water sweep efficiency. However, chemical-flooding technolo© XXXX American Chemical Society

Received: April 26, 2013 Revised: June 14, 2013 Accepted: June 25, 2013

A

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on the activity of chemolithotrophic microorganisms, there is little risk of inadvertently stimulating undesirable microbial processes, such as hydrocarbon degradation or sulfate reduction. Furthermore, both of the substrates of the NDFO organisms [Fe(II) and nitrate] and the various end products of the metabolism [Fe(III), nitrite, nitric oxide, and nitrous oxide] all have known anti-souring and sulfide-scouring activities.7

Table 1. Column Basics and the Running Conditions for the Dual-Column Experiment column basics fine grain size of glass beads (μm) 35−88 porosity (%)a 36.10 permeability (darcy)b 0.8 thickness of the beads bed (cm) diameter of the column (cm) flow rate (mL/min) running conditions

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MATERIALS AND METHODS Microorganism and Inoculation. The bacterial strain used in this study, Acidovorax ebreus,8 was isolated as a nitratedependent Fe(II) oxidizer. This organism oxidizes Fe(II) coupled to the complete reduction of nitrate according to 5Fe

2+

+

NO3−

+ 12H 2O → 0.5N2 + 5Fe(OH)3 + 9H

+

(1)

Sterile, anoxic, basal medium (BM) containing 4.7 mM NH4Cl, 0.5 mM Na2HPO3, and 1.3 mM KCl with the addition of vitamins and minerals outlined previously9 and pH buffered with 1,4-piperazinediethanesulfonic acid (PIPES) (10 mM, pH 7) was used for all experiments. The same medium pressurized (10 psi) by N2 was used as an influent to all columns. The sodium salt of phosphite was used in place of the original phosphate salt10 to prevent the formation of Fe(II) phosphate precipitates, which inhibit microbial iron oxidation. Flow Rate. The effluent from each column was collected by an individual polyvinyl chloride (PVC) pipe (40 mm inner diameter) supplied by McMasters-Carr equipped with a pressure transducer (Omega PX26 series) installed at the bottom. The pressure applied on the transducer increased linearly with an increasing height of the water column in the effluent collector, providing a continuous measurement of the cumulative effluent volume. The pressure change was recorded in real time by an eDaq e-corder (model ED1621). The flow rate in each column was calculated by taking the derivative of the volume versus time function. Fe(II) and Nitrate Analyses. Samples for iron analysis were extracted in 3 N HCl overnight. The iron concentrations were analyzed using the ferrozine assay as described elsewhere.11 Ion chromatography with conductivity detection (Ion Pac AS9-HC analytical column, Dionex DX-500 system, Dionex Corp., Sunnyvale, CA) was used to analyze nitrate and nitrite concentrations as previously described.10 Column Packing, Construction, and Disassembly. Sealed anaerobic column sets inoculated with active cultures of A. ebreus were constructed using 50 mL hypodermic glass syringes from Cadence Science (see SI Figure 1 of the Supporting Information). Glass beads of two different grain sizes were used to pack the individual columns in the dual column system (Table 1). Glass beads were acid-washed and rinsed with deionized (DI) water prior to mixing with DI water to create a slurry. The columns were wet-packed by scooping the bead slurry into the columns using a spatula. The column bed was tightly packed by regularly tapping the column to increase particle sedimentation and exclude gas bubbles. The dual-column system was set up as shown in SI Figure 1 of the Supporting Information. A glass wool pad was placed on the bottom of the bead beds to prevent beads from falling into the inlet tubing. The total void volumes of the “fine” and “coarse” columns were 30 and 32 mL, respectively. The influent media were continuously fed into the columns at 0.1 mL/min by a Dynamax Rainin peristaltic pump (model RP-1) providing a 3.5 h residence time inside the column. Seven pore volumes of

coarse ∼700 38.50 14.6 10 3 0.1

column sets

medium amendments

1 2 3 4 5

nitrate, Fe(II), acetate, and A. ebreus nitrate, Fe(II), acetate, and A. ebreus Fe(II), acetate, and A. ebreus nitrate, acetate, and A. ebreus nitrate, Fe(II), acetate, and heat-killed A. ebreus

a The porosity was obtained using the weight difference of a packed column filled with or without a liquid (water or methanol). bThe permeability was obtained by the falling-head method.29

anoxic BM were pumped through the columns to equilibrate the packing and remove any residual oxygen. The inlet tubing of the “coarse” column was partially clamped during this period to ensure that both columns in each set were flushed with an equal volume. Columns were inoculated with active cultures of A. ebreus grown anaerobically on acetate (6.25 mM) and nitrate (10 mM) in BM. The pump was shut down for 48 h, and 5 mL of growth culture was injected directly into the bottom port of each column. Acetate and nitrate were injected through the rubber stopper inserted in the top of the columns from aqueous stocks to give a final concentration of 10 mM. At the end of 48 h, the liquid layer on top of the bed of glass beads in all columns had become optically dense (OD600 = 0.8) because of cell growth. After initial inoculation and equilibration with anoxic medium, the columns were continuously fed with phosphite-based BM amended with nitrate and Fe(II) (10 mM each) as needed (Table 1) at a hydraulic residence time of about 3.5 h inside the column. Acetate (0.1 mM) was added as a suitable carbon source to maintain an active microbial population. A “heat-killed” control column set was prepared by placing the columns in a water bath at 95 °C for 2 h after the initial 48 h inoculation/incubation period. Some small gas bubbles formed in the columns during heating but were removed by tapping the columns gently when tilting the columns at different angles. The heated columns were tapped again vertically to make sure that the columns were as tightly packed as the rest of the columns. Samples for iron and nitrate analyses were taken directly from the liquid layer through the rubber stopper using a syringe rinsed by N2. After the columns were run for about 1 month, they were taken down for solidphase iron analysis. The columns were put in the freezer at −20 °C for 24 h. The frozen glass beads column could easily slip out from the glass syringe after being wrapped by a wet paper towel briefly. Each column was then sliced into four equal parts. Each part was added to 3 N HCl, and the liquid volume was brought up to 100 mL with 3 N HCl. Oil-Saturated Columns. Oil-saturated packed column sets were prepared in duplicate from 50 mL hypodermic glass syringes packed with different mesh size (mesh 50/70 and 170/ 325) glass beads, as outlined above. Motor oil (Ace All Season B

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quantity of precipitant produced was directly dependent upon the phosphate content of the medium, and in the presence of 5 mM phosphate, more than 87% of the initial 10 mM Fe(II) precipitated (data not shown). Although phosphorus is a prerequisite nutrient for all organisms on Earth, the environmentally dominant orthophosphate (PO43−) form is poorly soluble, especially in the presence of Fe(II).13 Furthermore, our recent studies have indicated that solid-phase Fe(II) is less bioavailable for NDFO by A. ebreus because this metabolism requires that Fe(II) enters the periplasmic space of the microbial envelope.12 In support of this, A. ebreus oxidized only ∼40% of the initial 10 mM FeCl2 added to the medium containing 0.5 mM phosphate after 50 h of incubation (see SI Figure 2a of the Supporting Information). In an effort to prevent uncontrolled abiotic precipitation of iron−phosphorus minerals, we replaced phosphate in BM with equimolar amounts of phosphite. Phosphite is ∼1000-fold more soluble than phosphate,14 and many organisms are capable of phosphite assimilation.15 In support of this, no abiotic Fe(II) precipitation was observed with phosphite, and growth with acetate and nitrate indicated that A. ebreus grew equally well in medium with either phosphate or phosphite as the phosphorus source (see SI Figure 2b of the Supporting Information). The ability of A. ebreus to assimilate phosphorus in the form of phosphite is explained by an analysis of the genome, which revealed the presence of homologues (IMG locus tags from Dtpsy_1417 to Dtpsy_1421) of the well-characterized ptxABCDE assimilatory phosphite uptake and oxidation system found in Pseudomonas stutzeri.1617 Furthermore, NDFO studies in phosphite BM indicated a significant improvement in both rate and extent of Fe(II) oxidation relative to phosphate BM, and almost 90% of the initial 10 mM FeCl2 was oxidized within 5 h by A. ebreus (see SI Figure 2a of the Supporting Information). Column Studies. To examine the effect of NDFO and solid-phase iron precipitation on fluid flow in a dynamic system, advective upflow column studies were performed. These studies used column sets each containing two columns of different permeabilities and joined by a common feed tube (see SI Figure 1 of the Supporting Information). In each instance, the total fluid flow through an individual column in a set was controlled by the relative permeability of the joined columns. In all column sets, regardless of treatment, 100% of the initial injection fluid flow passed through the high permeability column packed with the coarse grain glass beads (coarse columns) (Figure 2). However, within a short time frame (∼48 h), hydrological alteration indicative of permeability modification was observed and the flow change was specific to the treatment applied (Table 1). In the case of the heat-killed control column set, minimal fluid-flow alteration was observed throughout the column study (28 days), with effluent flow being almost exclusively from the coarse column (Figure 2e), indicating that any fluid flow alteration (permeability modification) observed in the treated columns was due to microbial metabolism. This was further supported by the flow results from the column set from which nitrate was omitted (Figure 2c). In this instance, the flow switched once from the coarse column to the low permeability column packed with fine grain glass beads (fine column) within the first 48 h of inoculation and equilibration. However, the flow quickly reverted exclusively back to the coarse column, where it remained for the duration of the column study (28 days) (Figure 2c). These results are in stark contrast to the columns

Motor Oil SAE 10W40) was pumped into each individual column at the inlet at 0.1 mL min−1 until the bed was saturated and the oil had reached the surface. The columns were then flushed with anoxic BM to flush out the oil. The produced fluids (oil/BM mix) from each column were collected in glass, graduated cylinders. About 2 g of NaCl was added to enhance separation of the oil and aqueous phases, and the recovered oil volume was observed. When no further oil was observed in the effluent, 5 mL of an active A. ebreus culture was injected directly into the bottom port of the columns. An equivalent volume (5 mL) of sterile anoxic DI water was injected into the control columns. All column sets were subsequently flooded with sterile anoxic BM containing 10 mM NaNO3 and 10 mM FeCl2 at 0.1 mL min−1. The volume of oil recovered from each column across all column sets was recorded daily.

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RESULTS NDFO Process. A. ebreus is a motile, Gram-negative facultative anaerobe previously isolated from the U.S. Department of Energy Integrated Field Research Challenge Site, Oak Ridge, TN.8 A. ebreus oxidized Fe(II) mixotrophically with acetate as an additional carbon and energy source, using nitrate as the sole electron acceptor (Figure 1).8,12 No Fe(II) was

Figure 1. Mixotrophic Fe(II) oxidation by A. ebreus with acetate as an additional carbon and energy source using nitrate as the sole electron acceptor.

oxidized in the absence of nitrate, and Fe(II) oxidation resulted in the production of equimolar amounts of an Fe(III)-rich orange precipitant presumably as ferric oxyhydroxide (data not shown). Previous studies on NDFO by A. ebreus indicated that a growth benefit is not observed from Fe(II) oxidation and growth may in fact be retarded by iron.12 Furthermore, those studies indicated that nitrite, nitrous oxide, and nitric oxide, all known inhibitors of microbial sulfate reduction, are produced during NDFO by A. ebreus at enhanced levels (as much as 10fold) relative to heterotrophic growth.12 In the current study, nitrite was detected in the effluent of column set 4 but was not found in the effluent of column sets 1 and 2 possibly because of the abiotic reaction with residual Fe2+. Neither nitrous oxide nor nitric oxide was analyzed as part of this study. Environmental Factors Controlling Fe(II) Solubility and NDFO Activity. Immediately after FeCl2 addition to freshly prepared anoxic basal medium, Fe2+ ions reacted, resulting in the abiotic production of a white precipitant. The C

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Figure 2. Change of the flow fraction through each column during the operation of the dual columns. The fraction of the flow in each column (a and b) in the presence of nitrate, Fe(II), and acetate, (c) in the presence of Fe(II) and acetate and absence of nitrate, (d) in the presence of nitrate and acetate and absence of Fe(II), and (e) heat killed in the presence of nitrate, Fe(II), and acetate. The dotted line at day 20 indicates a change in influent composition from the above media conditions to PIPES buffer only.

was transient and unstable (Figure 2d). The observed fluid dynamics in this case were probably due to slow growth and accumulation of the cells in the column matrix as a result of metabolism of the 0.1 mM acetate and nitrate in the medium. However, once acetate and nitrate were omitted at day 20, A. ebreus cells died and lysed, re-establishing the original flow path through the course column. Even so, similar biomass occlusion was unlikely to significantly control fluid dynamics in column sets 1 and 2, because Fe(II) is inhibitory to growth of A. ebreus.12 Accumulated Volume of Influent Passing through the Columns. The results outlined above demonstrate that NDFO can significantly alter hydrology in a dynamic system. This observation is more clearly seen in the cumulative volume flow analysis through the individual columns over the first 20 days of operation (Figure 3). In the case of the control column sets (heat killed or no nitrate addition), almost 100% of the accumulated liquid passed through the coarse column as expected (Figure 3). In contrast and despite the irregular flow switching between the two columns, the total liquid volume passing through each of the “fine” and “coarse” columns in the replicate sets 1 and 2 was almost identical by the end of the 20 day operation period (Figure 3), indicting effective flushing of both columns in each set.

containing an A. ebreus culture amended with nitrate and Fe(II) (column sets 1 and 2). In these replicate column sets, flow switching between the coarse and fine columns was observed sporadically within the initial 20 day operation period, indicating a continual permeability modification within the dynamic system (panels a and b of Figure 2). These results were reproducible in several separate renditions of this experimental regime (data not shown). After day 20, when injected medium was replaced with BM lacking Fe(II), nitrate, and acetate, limiting further microbial metabolism, fluid flow continued almost exclusively through the fine columns for the remainder of operation (panels a and b of Figure 2), suggesting that the permeability change in the coarse column matrix was stable. To account for the possibility of fluid occlusion and permeability alteration because of biomass accumulation and plugging rather than iron mineral precipitation, no Fe(II) was added to column set 4. In this instance, flow did not switch from the coarse column to the fine column until 8 days after inoculation and equilibration, from which point fluid flow altered several times prior to day 20 (Figure 2d). However, in contrast to column sets 1 and 2, when the injected medium was replaced with BM at day 20 [no addition of Fe(II), nitrate, or acetate], the flow switched back exclusively to the coarse column for the remainder of operation, indicating that the original permeability alteration because of biomass plugging D

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Figure 3. Cumulative volume of liquid that passed through the respective columns during the first 20 days of operation. In (a and b) columns amended with nitrate, Fe(II), and acetate, (c) columns amended with Fe(II) and acetate but not nitrate, (d) columns amended with nitrate and acetate but not Fe(II), and (e) heat-killed columns amended with nitrate, Fe(II), and acetate.

Retention and Distribution of Iron Minerals in the Columns. A comparison of inlet and outlet Fe(II) concentrations of the individual columns throughout the study indicated that significant quantities of iron [70−76% of total Fe(II) injected] were retained within each column in sets 1 and 2. Over the course of the 20 day treatment, a total of 11.0 and 10.2 mmol of iron were retained in sets 1 and 2, respectively, with an almost 50:50 (percentage of total iron) distribution across each column in set 1 and a 60:40 (percentage of total iron) distribution across the two columns in set 2, with the majority of the iron accumulating in the “coarse” column (Figure 4). A similar analysis in column sets 3 and 5 [both amended with Fe(II) but lacking either nitrate or active cells] indicated that no significant iron accumulation occurred (0.05 and 1.07 mmol for sets 3 and 5, respectively) (Figure 4), confirming the prerequisite participation of NDFO metabolism by A. ebreus in iron accumulation. Analysis of the iron distribution throughout the individual columns at the end of operation revealed that the iron minerals were not evenly precipitated along the length of the matrix. Rather, they were concentrated at the region closest to the column inlet (see SI Figure 3 of the Supporting Information), suggesting that NDFO activity primarily occurred near the inlet. Total and ferrous analyses identified the presence of both Fe(II) and Fe(III) in the solid phase, indicating mixed valence minerals (see SI Figure 3 of the Supporting Information). This is consistent with previous studies on NDFO using Azospira suillum, which demonstrated biogenesis of various mixed valence iron minerals, including green rust, maghemite, and magnetite.18 The total solid-phase iron recovered from the columns was consistent with the calculated amount retained equating to 6.6 and 7.0 mmol of Fe(III) and 3.2 and 2.4 mmol of Fe(II) for column sets 1 and 2, respectively. This indicates that 89.1 and 92.2% of the total iron removed from the liquid

phase in column sets 1 and 2, respectively, were recovered from the acid extracts. Using the density of iron hydroxide [Fe(OH)3] (4 g cm−3) as the average value for the biogenic minerals precipitated, the total volume of iron minerals formed during column operation was approximately 0.2−0.3 mL. The empirically determined porosity change of the treated column sets contributed by the biominerals is approximately 0.9−1.4%. The porosity change caused by biomass after inoculation and equilibration is negligible because of growth inhibition by Fe(II) to the A. ebreus cells and the minimal acetate level (0.1 mM) in the influent BM. However, the seemingly small change in overall porosity by the iron minerals was enough to divert the flow from the “coarse” column to the “fine” column in each set in a reproducible manner. This is most likely because the flow path is dictated by the relative differences in permeability between the individual columns in any column set rather than the total permeability of the columns as a whole. Because the majority of the mineral-phase iron precipitation occurred at the matrix around the inlet of the column, this is where the major permeability alteration occurred under the experimental regime tested. EOR from NDFO. To examine the effectiveness of NDFO on the EOR, oil-saturated column studies were performed. The columns were constructed identically to those outlined in SI Figure 1 of the Supporting Information, except that the packed matrix was saturated with motor oil (Ace All Season Motor Oil SAE 10W40) prior to being equilibrated with BM and inoculated. During the initial equilibration phase, an average of 45% (∼25 mL) of the total oil content of the individual column sets was flushed out and recovered, with the remaining 55% being retained within the column matrix and distributed across both the coarse and fine columns (panels a and b of Figure 5). No further oil was recovered from the control columns beyond this point, even after an extended flushing E

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Figure 4. Accumulated solid-phase iron retained (solid lines) in (a and b) column sets C1 and C2 amended with nitrate, Fe(II), and acetate, (c) column set C3 amended with Fe(II) and acetate but not nitrate, and (d) column set C5 heat killed and amended with nitrate, Fe(II), and acetate. The secondary y axis showed the flow change (dots) at the same time for better comparison of the change in iron retained.

hydrology of an advective flow system. In this instance, the flow regimes of two matrices of significantly different permeability were normalized to the extent that floodwater flow was distributed evenly across them. This was further supported by the significant enhancement (almost 100%) improvement in the total oil recovered from the system. In addition to the resultant improvement in sweep efficiency and oil recovery, NDFO can also benefit oil recovery processes through its imposed inhibition of microbial sulfate reduction and reservoir souring. The generation of hydrogen sulfide (H2S) as a metabolic end product of microbial sulfate respiration results in a variety of problems, including reservoir souring, crude oil contamination, metal corrosion, and metal sulfide precipitation that can plug pumping wells.19 Nitrate addition has been shown to impede SRB activity,20 and this is the basis of current technology aimed at controlling reservoir souring. Other oxyanions of nitrogen (NO2−, NO, and N2O) have an even more pronounced inhibitory effect on microbial sulfate reduction,21 and previous studies have clearly demonstrated enhanced production of these metabolites by NDFO microorganisms.12,22 Furthermore, the insoluble iron end products of NDFO will have scavenging capacity for H2S immobilizing it as iron sulfide (FeS) or pyrite (FeS2).

period of 18 days. In contrast, additional oil was recovered (22 and 12 mL) from the replicate column sets amended with A. ebreus, Fe(II), and nitrate. Additional oil recovery began within 2−4 days of inoculation of the column sets (panels a and b of Figure 5) and was a direct result of the hydrological flow alteration, shifting influent flow between the coarse and fine columns within the respective column sets. The total oil recovered over the 18 day operation from the treated replicate column sets was 72 and 88% of the initial oil in place (OIP), which represented a significant improvement in oil recovered relative to the untreated replicate column sets (42 and 48% of the OIP).

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DISCUSSION Our study suggests that NDFO has great potential for application in MEOR by improving the sweep efficiency and enhancing both the quality and quantity of oil recovered. Current limitations imposed on oil recovery relate to the heterogeneous nature of the subsurface reservoir rock matrix, resulting in irregular fluid flow pathways and poor sweep efficiencies of injected floodwaters. In this study, we provide evidence supporting the production of iron mineral precipitants through the stimulated NDFO, with resultant alteration of the F

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 510-643-8455. Fax: 510-642-4995. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for the investigation of MEOR was provided to John D. Coates by the Energy Bioscience Institute, University of California Berkeley/BP. The authors acknowledge Iain Clark for designing the signal conditioning circuit used for pressure measurements and Bernice Chu for assistance with sample preparation.

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Figure 5. (a) Volume and (b) percentage of oil recovered from duplicate inoculated test column sets (C1 and C2) amended with acetate, Fe(II), and nitrate, as compared to duplicate inoculated control column sets (C3 and C4) not amended with Fe(II) and nitrate.

The NDFO process is accompanied by the production of nitrogen gas. In our studies, we observed production of small bubbles of nitrogen gas inside the treated columns. Because of its miscibility in oil, N2 reduces crude oil viscosity, making it more inviscid. Furthermore, the in situ production of N2 increases the driving pressure in the reservoir, thus mobilizing the oil. Being cheap and non-corrosive, N2 has long been successfully used as the injection fluid for EOR and is widely used in oil field operations for reservoir pressure maintenance and gas lift by miscible gas displacement.23−25 Under higher pressure in the oil reservoir, nitrogen can form a miscible slug, which aids in mobilizing the oil from the reservoir rock.26−28

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REFERENCES

(1) Fisher, W. L.; Tyler, N.; Ruthven, C. L.; Burchfield, T. E.; Pautz, J. F. An assessment of the oil resource base of the United States. Oil Resources Panel; Bartlesville Project Office: Bartlesville, OK, 1992. (2) Youssef, N.; Elshahed, M. S.; McInerney, M. J. Chapter 6 Microbial processes in oil fields: Culprits, problems, and opportunities. Adv. Appl. Microbiol. 2008, 66, 141−251. (3) Green, D. W.; Willhite, G. P. Enhanced Oil Recovery; Society of Petroleum Engineers: Richardson, TX, 1998. (4) Strand, S.; Standnes, D. C.; Austad, T. Spontaneous imbibition of aqueous surfactant solutions into neutral to oil-wet carbonate cores: Effects of brine salinity and composition. Energy Fuels 2003, 17, 1133− 1144. (5) Weihong, Q. D.; Liangjun, D.; Zhongkui, Z.; Jie, Y.; Huamin, L.; Zongshi, L. Interfacial behavior of pure surfactants for enhanced oil recoveryPart 1: A study on the adsorption and distribution of cetylbenzene sulfonate. Tenside, Surfactants, Deterg. 2003, 40, 87−89. (6) Weber, K. A.; Achenbach, L. A.; Coates, J. D. Microorganisms pumping iron: Anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 2006, 4 (10), 752−764. (7) Gieg, L. M.; Jack, T. R.; Foght, J. M. Biological souring and mitigation in oil reservoirs. Appl. Microbiol. Biotechnol. 2011, 92 (2), 263−282. (8) Byrne-Bailey, K. G.; Weber, K. A.; Chair, A. H.; Bose, S.; Knox, T.; Spanbauer, T. L.; Chertkov, O.; Coates, J. D. Completed genome sequence of the anaerobic iron-oxidizing bacterium Acidovorax ebreus strain TPSY. J. Bacteriol. 2010, 192 (5), 1475−1476. (9) Bruce, R. A.; Achenbach, L. A.; Coates, J. D. Reduction of (per)chlorate by a novel organism isolated from paper mill waste. Environ. Microbiol. 1999, 1 (4), 319−329. (10) Weber, K. A.; Pollock, J.; Cole, K. A.; O’Connor, S. M.; Achenbach, L. A.; Coates, J. D. Anaerobic nitrate-dependent iron(II) bio-oxidation by a novel, lithoautotrophic, betaproteobacterium, strain 2002. Appl. Environ. Microb. 2005, 72, 686−694. (11) Lovley, D. R.; Phillips, E. J. P. Availability of ferric iron for microbial reduction in bottom sediments of the fresh-water tidal Potomac River. Appl. Microbiol. Biotechnol. 1986, 52, 751−757. (12) Carlson, H. K.; Clark, I. C.; Blazewicz, S. J.; Iavarone, A. T.; Coates, J. D. Fe(II) oxidation is an innate capability of nitrate-reducing bacteria involving abiotic and biotic reactions. J. Bacteriol. 2013, 195, 3260−3268. (13) Pasek, M. A.; Kee, T. P.; Bryant, D. E.; Pavlov, A. A.; Lunine, J. I. Production of potentially prebiotic condensed phosphates by phosphorus redox chemistry. Angew. Chem., Int. Ed. 2008, 47, 7918− 7920. (14) Pasek, M. Rethinking early Earth phosphorus geochemistry. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 853−858. (15) White, A.; Metcalf, W. Microbial metabolism of reduced phosphorus compounds. Annu. Rev. Microbiol. 2007, 61, 379−400. (16) Martinez, A.; Osburne, M. S.; Sharma, A. K.; DeLong, E. F.; Chisholm, S. W. Phosphite utilization by the marine picocyanobacte-

ASSOCIATED CONTENT

S Supporting Information *

In a dynamic system, advective upflow column studies were performed using column sets containing a high- and lowpermeability packing and joined by a common feed tube (SI Figure 1), effects of phosphorous species on (a) rate and extent of Fe(II) biooxidation and (b) growth of A. ebreus (SI Figure 2), and distribution of iron minerals along the length of columns in (a) columns amended with nitrate, Fe(II), and acetate (C1 and C2) and (b) columns amended with Fe(II) and acetate but no nitrate (C3) and heat-killed columns amended with nitrate, Fe(II), and acetate Fe(II) (C5) (SI Figure 3). This material is available free of charge via the Internet at http://pubs.acs.org. G

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dx.doi.org/10.1021/es401838b | Environ. Sci. Technol. XXXX, XXX, XXX−XXX