Microbially Enhanced Oil Recovery by Sequential Injection of Light

Sep 25, 2015 - Heavy oil from the MHGC field near Medicine Hat, Alberta, Canada ..... We thank Dr. Rhonda Clark for administrative support and Drs. Yu...
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Microbially Enhanced Oil Recovery by Sequential Injection of Light Hydrocarbon and Nitrate in Low- And High-Pressure Bioreactors Fatma Gassara, Navreet Suri, Paul Stanislav, and Gerrit Voordouw* Petroleum Microbiology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada S Supporting Information *

ABSTRACT: Microbially enhanced oil recovery (MEOR) often involves injection of aqueous molasses and nitrate to stimulate resident or introduced bacteria. Use of light oil components like toluene, as electron donor for nitratereducing bacteria (NRB), offers advantages but at 1−2 mM toluene is limiting in many heavy oils. Because addition of toluene to the oil increased reduction of nitrate by NRB, we propose an MEOR technology, in which water amended with light hydrocarbon below the solubility limit (5.6 mM for toluene) is injected to improve the nitrate reduction capacity of the oil along the water flow path, followed by injection of nitrate, other nutrients (e.g., phosphate) and a consortium of NRB, if necessary. Hydrocarbon- and nitrate-mediated MEOR was tested in low- and high-pressure, water-wet sandpack bioreactors with 0.5 pore volumes of residual oil in place (ROIP). Compared to control bioreactors, those with 11−12 mM of toluene in the oil (gained by direct addition or by aqueous injection) and 80 mM of nitrate in the aqueous phase produced 16.5 ± 4.4% of additional ROIP (N = 10). Because toluene is a cheap commodity chemical, HN-MEOR has the potential to be a cost-effective method for additional oil production even in the current low oil price environment.

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INTRODUCTION Oil production often involves water injection to maintain reservoir pressure and sweep oil from injection to producing wells. Following water breakthrough, the latter will produce a mixture of oil and water of which the oil fraction decreases with time. Early water breakthrough in low temperature oil fields, such as the Medicine Hat Glauconitic C (MHGC) field in Alberta, which produces viscous heavy oil, is caused by the large difference in viscosity between the residual oil in place (ROIP) and the injected water. Once water breakthrough by “viscous fingering” occurs, the water tends to travel established paths of least resistance, decreasing oil production. Enhanced oil recovery (EOR) methods can include the amendment of injection water with polymers to increase viscosity. Extracting more oil from reservoirs through EOR methods is a major challenge to the oil industry.1−3 MEOR uses indigenous or injected microbes to improve the recovery of ROIP from depleted reservoirs.1−3 These ferment inexpensive raw materials such as molasses to desired products, for example, organic acids, alcohols, gases, biomass, biopolymers, and/or biosurfactants.1−5 Although the high solubility of molasses in water and its insolubility in oil would seem advantageous for MEOR applications, this also means that molasses is removed from the reservoir by water injection. The use of oil-soluble, low molecular weight MEOR substrates, like toluene or heptane, could be advantageous in this respect, as these bind to oil preventing them from being lost during continued water injection. © XXXX American Chemical Society

MEOR can also involve injection of nitrate, a high-potential electron acceptor for heterotrophic nitrate-reducing bacteria (hNRB) in oil fields.1,4,6,7 These use alkylbenzenes, especially toluene, as the preferred electron donor.8,9 Production of heavy oil from sand pack bioreactors increased by the injection of nitrate,10 but may have been limited by the low concentration of toluene in heavy MHGC oil of 1−2 mM.8,9 In the present study we explore whether increasing the concentration of toluene or of toluene and heptane in MHGC oil increases nitrate reduction and the associated production of heavy oil.

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MATERIALS AND METHODS Toluene-Oxidizing Nitrate-Reducing Enrichment Cultures. Heavy oil from the MHGC field near Medicine Hat, Alberta, Canada with an American Petroleum Institute (API) gravity of 16° and a viscosity of 3400 cP at 20 °C was used. The MHGC field is a shallow (850 m), low-temperature (30 °C) field from which heavy oil is produced by water injection. The field is subject to nitrate injection to limit microbial H2S production and produced waters contain a significant fraction of the hNRB Thauera.9,11 Enrichment cultures of hNRB were grown in 120 mL serum bottles, containing 47.5 mL of sterile anaerobic CSBK medium (Table S1, Supporting Information) Received: August 12, 2015 Revised: September 22, 2015 Accepted: September 25, 2015

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DOI: 10.1021/acs.est.5b03879 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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each of three experiments two or four up-flow stainless steel bioreactors (17.5 × 2 or 35 × 2 cm) were packed with 140− 200 mesh sand and flooded with CSBK medium at high pressure (400 psi, 27.2 atm) to decrease formation of gaseous N2 and CO2. Bioreactors (PV of 17.5 or 35 mL) were then flooded with 1 PV of heavy oil or with 1 PV of heavy oil with 11.4 mM of toluene, followed by flooding with CSBK to residual oil in stage 1. This was followed by injection of 0.5 PV of hNRB suspended in CSBK with or without 80 mM nitrate and incubation for 14 days without flow and under pressure in stage 2. Flow was then resumed at 1 PV/day in stage 3 by injecting CSBK. Effluent fractions were collected to determine produced oil and its concentration of toluene, as well as the aqueous concentrations of nitrate and nitrite. Increasing the Toluene Concentration of ROIP by Injecting Aqueous Toluene. Bioreactors were injected with CSBK containing up to 3 mM of dissolved toluene, as indicated in the Results section. Increased concentrations of toluene in produced or residual oil were determined from the OD600 of the DCM extracts and their toluene concentration as determined by GC-MS. Quantification of Toluene in Oil by Gas Chromatography−mass Spectrometry (GC-MS). After measuring the oil content of the produced oil−water mixtures, 1 mL of each oil−DCM phase was amended with 1 μL of internal standard (deuterated toluene: toluene-d8: purity >99.9%; Sigma-Aldrich, Canada). Aliquots (1 μL) of these were then injected by an autoinjector (7683B series, Agilent Technologies) into a GC (7890N series, Agilent Technologies), which was connected to a mass-selective detector (5975C inert XL MSD series, Agilent Technologies). The GC was equipped with an HP-1 fused silica capillary column (length 50 m, inner diameter 0.32 mm, film thickness 0.52 μm; J&W Scientific). Helium was used as the carrier gas. Peaks of interest were identified from their mass spectra using the Wiley registry. The concentration of toluene in MHGC oil was calculated from its peak area relative to that of the internal standard. Chemical and Physical Analyses. Bioreactor effluent (1 mL) was centrifuged and the supernatant used for the assay of nitrate and nitrite by HPLC using a UV detector (Gilson, Lewis Center, OH), as indicated elsewhere.9 The emulsification index (E24) of centrifuged bioreactor effluents was determined by adding 2 mL of toluene to 2 mL of effluent in a 10 mL test tube. After vortexing for 2 min the tubes were left to stand for 24 h and E24 (%) was calculated as 100*(height of the emulsified layer/total height of the liquid column). The pressure difference (ΔP) between the inlet and the outlet was determined using a digital pressure transmitter (Siemens Sitrans PDS III, USA) for high or a digital manometer (Dwyer Series 490, Michigan City, IN) for low pressure bioreactors. The relationship between flow rate through the porous medium q (cm3s−1), permeability K (Darcy), cross-section area A (cm2), pressure drop ΔP (atm), path length L (cm) viscosity η (cP) and permeability is given by the Darcy equation

with up to 80 mM NaNO3, 1 mL of MHGC oil, a headspace of 90% (v/v) N2 and 10% CO2 (N2−CO2), and additional electron donors, as described in Table S2. The bottles were inoculated with 2.5 mL of MHGC produced water and incubated at 30 °C. MHGC oil (1 mL) was amended with up to 570 mM of toluene to reduce up to 80 mM of nitrate in 47.5 mL of aqueous phase. When using bioreactors with 0.5 pore volume (PV) of residual oil a toluene concentration of 11.4 mM was used in the oil phase (61 μL of toluene per 50 mL of oil) to reduce 80 mM nitrate in 0.5 PV of aqueous phase. Samples from enrichment cultures were taken with N2−CO2 flushed syringes to measure nitrate and nitrite concentrations with high performance liquid chromatography (HPLC). Low Pressure Bioreactors. Syringes of 30 mL without piston were packed with glass wool, polymeric mesh and then with sand (Sigma-Aldrich, 50−70 mesh), followed by glass wool.10,12 A rubber stopper perforated with a syringe needle was used to seal the columns. Zip ties were used to enhance the seal (Figure S1). Luer-Lock three-way valves were connected to the bottom syringe inlet and the needle outlet. These were connected to 0.76 mm ID PVC tubing (Mandel Scientific) with the aid of steel fittings (Ochs Laborbedarf). The influent tubing was connected to both sides of a piece of calibrated 0.5 mm ID PVC pump tubing (Mandel Scientific), placed in the head of an 8-channel peristaltic pump (Gilson Inc., Minipuls-3), through 1 mm OD steel connectors (Gilson Inc.). CSBK medium was pumped from sealed bottles with an N2−CO2 headspace. The effluent was led into perforated Falcon tubes. Up to six columns were up-flow injected and weighed before and after water saturation to measure PV, which ranged from 14 to 17 mL. The columns were then flooded with 1 PV of heavy oil amended with 0−11.4 mM of toluene or with heavy oil amended with 6 mM of heptane and 6 mM toluene, replacing 0.95 PV of water by oil. Oil was then produced by injection of anoxic CSBK at a rate of 1 PV/day. The oil content of the produced oil−water mixture was determined daily until a total of 0.5 PV of oil was produced (Stage 1, typically 2 weeks). Oil content was determined by adding a known volume of dichloromethane (VDCM), measuring the volume of water (VH2O) and then the volume of oil (Voil) by determining the optical density at 600 nm (OD600) of the oil−DCM phase to derive the concentration of oil (Coil) in mg/mL; OD600 was determined using a Thermo Scientific GENESY 20 spectrophotometer placed in a fume hood. Voil was then calculated as Voil = Coil × VDCM/ρoil, where ρoil is the density of MHGC oil (0.959 g/mL). In stage 2 columns were injected with nitrate and with microbial cultures grown as indicated in Table S2. These were transferred into 50 mL Falcon tubes, which were centrifuged for 20 min at 13 000 rpm. The biomass pellet was then suspended in 0.5 PV of anoxic CSBK medium with 0−80 mM of sodium nitrate and injected into the columns using the peristaltic pump. The columns were then sealed at the top end, while the bottom ends were attached to 60 mL vials through 18G effluent port needles (Figure S2). Vials were changed regularly to measure produced water and oil over 14 days (stage 2), when no further fluid production was observed. Following incubation, up-flow injection of CSBK medium was resumed at 1 PV/day in stage 3. Concentrations of nitrate and nitrite in the effluent aqueous phase and of toluene in the effluent oil phase were measured by HPLC and GC-MS, respectively. High Pressure Bioreactors. Packing and operating of high-pressure bioreactors are described in Appendix 1. A schematic of the experimental set up is shown in Figure S3. In

q = −(K *A*ΔP)/(η*L)

(1)

Microbial Community Analysis. This was done as indicated elsewhere13 and in Appendix 1 of the Supporting Information. Data analysis was conducted with Phoenix 2.14 The entire set of raw reads is available from Sequence Read Archive (SRA) at NCBI under accession number SRX1084994. B

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Environmental Science & Technology Table 1. Oil Production (mL and % ROIP) from Low Pressure Bioreactors with PV = 15 mLa bioreactor

electron donor (mM)

nitrate (mM)

ROIP (ml)

Bio_I1 Bio_II1 Bio_III1 Bio_IV1 Bio_IV2 average ± SD Bio_I6 Bio_II4 Bio_IV6 Bio_VI5 Bio_VI6 average ± SD

toluene (0) toluene (0) toluene (0) toluene, heptane (6,6) toluene, heptane (6,6)

0 0 0 0 0

6.88 8.34 6.16 7.32 7.09

oil (mL) stage 2 0 0 0 0 0

toluene (11.4) toluene (11.4) toluene, heptane (6,6) toluene (11.4) toluene (11.4)

80 80 80 80 80

6.26 8.12 7.66 7.29 6.91

1.48 1.13 0.87 1.18 1.26

oil (% ROIP) stage 2

oil (ml) final

oil (% ROIP) final

0.0 0.0 0.0 0.0 0.0 0.0 ± 0.0 23.6 13.9 11.4 16.2 18.2 16.7 ± 4.7

0.424 0.55 0.30 0.52 0.47

6.27 6.6 4.9 7.1 6.6 6.3 ± 0.8 28.91 18.1 23.8 21.9 22.1 23.0 ± 3.9

1.81 1.47 1.82 1.6 1.53

a

These contained the indicated concentrations of electron donor in the oil phase and of nitrate electron acceptor in the aqueous phase. Average oil production following stage 2 incubation or following final stage 3 elution are also shown.

Table 2. Oil Production (mL and % ROIP) in High-Pressure Bioreactors Containing the Indicated Concentrations of Toluene in the Oil Phase and of Nitrate in the Aqueous Phase Following Stage 2 Incubation and Stage 3 Elution (Oil Final)

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bioreactor

toluene (mM)

nitrate (mM)

PV (mL)

ROIP (mL)

ROIP (PV)

oil (mL) final

oil (% ROIP) final

Bio_VII1 Bio_VIII1 Bio_IX1 average ± SD Bio_VII2 Bio_VIII2 Bio_IX2 average ± SD

0 11.4 11.4

0 0 0

34 17.5 36.2

12.9 7.85 15

0.92 0.35 0.76

11.4 11.4 11.4

80 80 80

34.8 17.5 34.9

13.2 8.03 14.7

0.38 0.45 0.41 0.41 ± 0.4 0.38 0.46 0.42 0.42 ± 0.4

7.1 4.5 5.1 5.6 ± 1.4 17.7 19.7 16.3 17.9 ± 1.7

RESULTS MEOR with Oil-Soluble Electron Donors in LowPressure Bioreactors. Experiments were conducted with 20 low-pressure bioreactors Bio_I1 to Bio_IV6, as indicated in Table S3. Results for a subset of 10 bioreactors with either 0 or 11.4 mM of toluene or 6 mM toluene and 6 mM heptane in the oil phase and of 0 or 80 mM nitrate in the aqueous phase are summarized in Table 1. Stage 2 incubation of Bio_I1 and Bio_I6 with 0 and 80 mM nitrate, respectively, gave no oil or water for Bio_I1, but did give oil (1.48 mL; 23.6% of ROIP) and water (3.97 mL) for Bio_I6 (Figure S4A; Table S3). Production of more water than oil may be caused by the fact that it is easier for produced N2 and CO2 to push water out of the columns than more viscous heavy oil. The final oil production at the end of stage 3 was 6.3 and 28.9% of ROIP Bio_I1 and Bio_I6, respectively (Figure S4A, Table 1), indicating that the use of nitrate during incubation increased oil production. Likewise, incubation of bioreactors Bio_II1 and Bio_II4 with an hNRB consortium and 0 or 80 mM nitrate and subsequent stage 3 elution gave 6.6 and 18.1% of ROIP, respectively (Tables 1 and S3). When bioreactors Bio_III1 and Bio_III4, containing 0.5 PV of oil not amended with toluene, were incubated with hNRB and 0 or 80 mM nitrate and then eluted with CSBK, nitrate was eluted from Bio_III4 with a peak concentration of 70 mM (Figure S5). The peak nitrite concentration was 4.5 mM (not shown). Following continued elution with CSBK a total of 4.9 and 6.25% of ROIP were produced, respectively (Figure S4B, Tables 1 and S3). Hence, when no toluene was added to MHGC oil, little nitrate was reduced and little oil was produced during stages 2 and 3. Likewise, when toluene was added to heavy oil but no nitrate was added to the aqueous phase no

2.34 1.58 2.39

additional oil production was observed (Table 2: Bio_VIII1 and Bio_IX1). This indicates that additional production of heavy MHGC oil requires the presence of both added toluene in the oil and of nitrate in the aqueous phase. Because heptane, which was below detection in the MHGC oil used, can also serve as an electron donor for nitrate reduction10 we used MHGC oil amended with 6 mM toluene and 6 mM heptane in bioreactors Bio_IV1 and Bio_IV6 (Figure S4C). The same, centrifuged hNRB consortium, grown with 80 mM nitrate and MHGC oil with toluene and heptane (Table S2), was then injected together with 0 or 80 mM nitrate into Bio_IV1 and Bio_IV6 for stage 2 incubation. Final oil production following stage 3 was 7.1 and 23.8% of ROIP for Bio_IV1 and Bio_IV6, respectively (Tables 1 and S3). Toluene quantitation indicated that the toluene concentration of MHGC oil decreased to zero in Bio_IV6 during stage 2 incubation, but not in Bio_IV1 (Figure S6A). HPLC analyses indicated that only 3 mM nitrate was left at the end of the stage 2 incubation of Bio_IV6 (Figure S6B). Overall production with high toluene or high toluene and heptane and 80 mM nitrate was 23.0 ± 3.9% of ROIP (N = 5), whereas the average was 6.3 ± 0.8% of ROIP (N = 5) with 0 mM nitrate (Table 1). This difference was statistically significant (p > 99%; student’s t test). MEOR with Aqueous Electrons Donors in LowPressure Bioreactors. Oil production using aqueous electron donors, is indicated in Table S3 and has been described elsewhere.15 In the presence of sufficient molasses, glucose or acetate to reduce 80 mM of nitrate, the average production was 25.0 ± 11.1% of ROIP (N = 5), similar to that obtained with high toluene or high toluene and heptane. C

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Figure 1. (A, D) Production of toluene-spiked heavy oil in PV plotted against the total volume of produced oil and water (PV) flooded through high-pressure bioreactors. The flow rate was 1 PV/day; 17.5 mL/day for (A) and 35 mL/day for (D). Bioreactors were eluted with CSBK in stage 1, incubated for 2 weeks in stage 2 with hNRB and either 0 or 80 mM of nitrate and again eluted with CSBK in stage 3. (B, E) Residual concentration of toluene (mM) in the produced oil. (C, F) Concentrations of nitrate and nitrite in effluents from Bio_VIII2 and Bio_IX2, which were incubated with 80 mM nitrate. No data are shown for Bio_VIII1 and Bio_IX1, which were incubated with 0 mM nitrate.

Toluene- and Nitrate-Mediated MEOR in HighPressure Bioreactors. Toluene- and nitrate-mediated oil production in low-pressure bioreactors may be driven in part by the formation of N2−CO2 gas. Since less gas will form under the high-pressure conditions existing in oil reservoirs, experiments were also performed at 400 psi (27.2 atm), under conditions indicated in Table 2. Oil production is shown in Figure 1A, D. Following stage 1 elution bioreactors contained on average 0.42 PV of MHGC oil (Table 2). For stage 2 incubations the same hNRB consortium was used for all bioreactors (Table S2). This was centrifuged to remove residual nitrate and then resuspended in CSBK without nitrate for Bio_VII1, Bio_VIII1, and Bio_IX1 and in CSBK with 80 mM nitrate for Bio_VII2, Bio_VIII2, and Bio_IX2 (Table 2). No oil was produced during stage 2 incubations, because the steel bioreactors were kept closed and under pressure. Following stage 3 flooding bioreactors Bio_VII1, Bio_VIII1, and Bio_IX1 produced 5.6 ± 1.4% of ROIP, irrespective of the absence (Bio_VII1) or presence (Bio_VIII1 and Bio_IX1) of toluene in the oil (Table 2). In contrast, Bio_VII2, Bio_VIII2, and Bio_IX2 produced 17.9 ± 1.7% of ROIP (Table 2). The difference was statistically significant (p > 99%; student’s t test), indicating that the presence of nitrate increased production by 12.3 ± 2.2%. The presence of nitrate caused toluene in the oil to be oxidized in Bio_VII2 and Bio_IX2 (Figure 1 B, E).

Toluene oxidation was coupled to nitrate reduction with 10−17 mM of nitrate and 2−5 mM nitrite remaining (Figure 1C, F). In the absence of nitrate no toluene oxidation was observed in Bio_VII1 and Bio_IX1 (Figure 1B, E). Retention of Aqueous Toluene Injected into OilContaining Bioreactors. The distribution of toluene between oil and aqueous phases is governed by the partition coefficient π = Co/Cw, where Co and Cw are the equilibrium concentrations of toluene in the oil phase and in water, respectively. A numerical value for π can be calculated from the solubility of pure toluene in water, that is, the concentration of toluene in the pure liquid (9442 mM) and its solubility in water at 20 °C (5.6 mM) give π = 1686. Values for π of toluene for MHGC oil and water were determined by adding toluene to either the oil or the aqueous phase and mixing for 1 to 7 days at 20 °C. This gave π = 17 800 ± 3000 and π = 1380 ± 420 (N = 2), respectively, indicating that equilibrium had not yet been reached under these conditions. Nevertheless, these values indicate that if aqueous toluene is injected slowly most would be expected to be retained by the oil. Low pressure bioreactors, containing 0.5 PV of residual MHGC oil as in Figure 1, were injected with 2 PV of CSBK containing Cw = 3 mM toluene at a flow rate of either 1.0 PV/ day (15 mL/day) or 0.5 PV/day (7.5 mL/day). The bioreactors were then cut and oil was extracted from five sections, D

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Figure 2. Oil production by sequential injection of toluene and nitrate in low-pressure bioreactors with PV of 15 mL. Bio_X1, Bio_X2, and Bio_X3 (Bio_X1−3) were not and Bio_X4, Bio_X5, and Bio_X6 (Bio_X4−6) were injected with toluene. All were injected with nitrate and hNRB in stage 2. (A) Average production of oil for Bio_X1−3 and Bio_X4−6. Standard deviation is shown when this exceeded the size of the symbols. (B) Differential pressure ΔP (mBar) for Bio_X1 and Bio_X6. Left scale is for data from PV 0 to 35. Right scale (inset) is for data from PV 22 to 35.

Sequential Injection of Toluene and Nitrate in High Pressure Bioreactors. Stainless steel, high-pressure bioreactors Bio_XI1, Bio_XI2, Bio_XII1, and Bio_XII2 were packed, injected with CSBK and then with 1 PV of oil without additional toluene, except Bio_XI1, which was injected with 1 PV of oil spiked with 11.4 mM toluene. All were flooded with 15 PV of CSBK to 0.45 PV of residual oil. Bio_XI2 and Bio_XII2 were then flooded with 10 PV of CSBK with 3 and 2 mM toluene, respectively, whereas Bio_ XI1 and Bio_XII1 were flooded with 10 PV of CSBK at 1 PV/day throughout. All bioreactors were injected with 0.5 PV of hNRB culture and 80 mM nitrate and incubated for 14 days after which flow of CSBK was resumed. Oil production is shown in Figure 3A, D. Bio_XI2 and Bio_XII2, injected with 3 and 2 mM toluene, produced an additional 36.5% and 23.4% of ROIP, respectively. Bio_XI1, flooded with oil spiked with 11.4 mM toluene, produced an additional 12.0% of ROIP, whereas Bio_XII1, containing oil without additional toluene, produced 8.5% of ROIP (Table S5). Effluents of Bio_XI1, Bio_XI2 and Bio_XII2 had a low, whereas effluent of Bio_XII1 (containing oil without added or injected toluene) had a high nitrate concentration (Figure 3 C, F). The pressure difference (ΔP) between the inlet and the outlet of Bio_XI2 and Bio_XII2 decreased from 3000 to 4000 mbar to 0−10 mbar but increased by 30 mbar at the start of stage 3 elution (Figure 3B, E). These increases in ΔP were similar as observed for low-pressure bioreactors (Figure 2B). In view of the large difference in oil production between Bio_XI1 (12%) and Bio_XI2 (36.5%), the emulsification index E24 (%) was measured for effluents from these bioreactors. E24(%) was found to be 3- to 6-fold larger for Bio_XI2 than for Bio_XI1, as indicated in Figure S8. Microbial Community Analysis. Microbial community analyses were done for hNRB cultures grown with different electron donors and 80 mM nitrate as electron acceptor at 30 °C. Following DNA isolation, PCR of 16S rRNA genes and pyrosequencing, similar numbers of quality-controlled reads were obtained (Table S6). At the genus level dominant community members following growth on toluene, sucrose, acetate, molasses and glucose included the hNRB Pseudomonas (1.4, 3.8, 8.1, 38, and 80%, respectively) and Thauera (95, 2.4, 3.5, 10.7, and 3.5%, respectively). Hence, Thauera dominated the community grown with toluene, as found in previous enrichment cultures and in the field.9

representing the entire column. Prior to toluene injection the toluene concentration in MHGC oil was 1.2 ± 0.3 mM (N = 2). Hence, if all toluene were to be retained Co would increase to 13.2 mM. Following injection at 1 PV/day the volume weighted Co increased to 3.1 mM, whereas at 0.5 PV/day this increased to 5.2 mM, representing increases of 1.9 and 4.0 mM, respectively. Based on these data we decided to inject 10 PV of CSBK with 3 mM toluene at 1 PV/day to achieve a Co of approximately 10 mM. However, note that because injected aqueous toluene is transferred from the aqueous phase into a viscous oil phase where diffusion is slow, its concentration in volume elements lining aqueous channels may be higher than the calculated average Co. Sequential Injection of Toluene and Nitrate in Low Pressure Bioreactors. Low-pressure bioreactors Bio_X1 to Bio_X6 were injected with 14 PV of CSBK to produce approximately 0.5 PV of oil. Bio_X1, Bio_X2, and Bio_X3 were then injected with 10 PV of CSBK, whereas Bio_X4, Bio_X5, and Bio_X6 were injected with 10 PV of CSBK with 3 mM toluene. The flow rate was 1 PV/day throughout. All bioreactors were then injected with 0.5 PV of hNRB culture with 80 mM nitrate and incubated for 14 days after which flow of CSBK medium at 1 PV/day was resumed (Figure 3A). This resulted in production of 3.0 ± 1.7% of ROIP from Bio_X1, Bio_X2 and Bio_X3 and of 20.6 ± 6.8% of ROIP from Bio_X4, Bio_X5, and Bio_X6 (Table S4). Hence, sequential injection of toluene and nitrate gave an additional 17.6 ± 7.0% of ROIP. Effluents of Bio_X4, Bio_X5, and Bio_X6 had a low, whereas effluents of Bio_X1, Bio_X2, and Bio_X3 had a high nitrate concentration of 70 mM (results not shown). Monitoring the pressure difference (ΔP) between the inlet and the outlet indicated a large drop from 3000 to 10 mbar in both Bio_X1 and Bio_X6 during stage 1 oil production (Figure 3B). Following stage 2 incubation, a transient increase in ΔP from 14 to 40 mbar was observed in Bio_X6, but not in Bio_X1 (Figure 3B). The relation between flow rate q and ΔP for sand pack bioreactors without and with 0.5 PV of residual oil is given in Figure S7, indicating that these have permeabilities of 1.63 and 0.72 Darcy. The difference indicates a 2.3-fold decreased area A for water flow in the sand pack with residual oil. The calculated ΔP for this sand pack to maintain a flow rate of 1 PV/day (15.7 mL/day, 0.0109 mL/min) is 0.65 mbar (Figure S7). The increase in ΔP from 14 to 40 mbar in Bio_X6 to maintain this flow rate can be interpreted in terms of the Darcy equation, as a 2.9-fold decrease in A, a 2.9-fold increase in viscosity η, or a combination of these two. E

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Figure 3. Oil production by sequential injection of toluene and nitrate in high-pressure bioreactors. (A, D) Bio_XI1 was injected with oil with 11.4 mM toluene, whereas Bio_XI2, Bio_XII1, and Bio_XII2 were injected with oil only. Following injection of 14 PV of CSBK, Bio_XI1, and Bio_XII1 were injected with another 10 PV of CSBK, wereas Bio_XI2 and Bio_XII2 were injected with 10 PV of CSBK with 3 and with 2 mM toluene, respectively. All bioreactors were incubated with 80 mM nitrate and the same hNRB consortium in stage 2, after which all were flooded with CSBK in stage 3. Production of oil in PV (1 PV = 35 mL) is plotted against the total volume of produced oil and water in PV. (B, E) Differential pressure ΔP (mBar) for Bio_XI2 and Bio_XII2. Left scale, ●; right scale, ○. (C, F) Concentration of nitrate for bioreactors, as indicated.

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DISCUSSION We have shown that additional oil can be produced from heavy oil-containing bioreactors in which hNRB activity is stimulated by the presence of added or injected oil-soluble electron donor (e.g., toluene) and aqueous nitrate. Oxidation of 11.4 mM toluene in the oil phase to CO2 and H2O requires 80 mM of nitrate in the aqueous phase:

fermenting bacteria (if a specific fermentation outcome is desired) is injected together4 and reaction will start as soon as all components are mixed prior to injection. The mechanisms of MEOR have been reviewed extensively.1,2,5,15,17 This includes the ability of bacteria to produce biosurfactants, decreasing interfacial tension of oil held in small rock pores.1,2,18 Biosurfactant production by injected Bacillus species, which were fed by coinjection of glucose, nitrate and inorganic nutrients, has been demonstrated in a field trial.4 However, the ability of bacteria to produce sufficient biosurfactants in situ has been questioned by Gray et al. due to significant adsorption to reservoir rock.17 These authors indicated that stimulation of surface-active bacteria producing oil emulsion droplets is a more likely mechanism. Blockage of preferred flow paths by in situ production of biomass and exopolysaccharide or other biopolymers is also thought to be an important MEOR mechanism.1,16,17,19 Formation of emulsified oil may also contribute to blockage,20,21 which is expected to increase the pressure gradient needed to maintain flow. Belayev et al.16 indicated a 1500 mbar increase in pressure during well stimulation in the Sernye Vody oilfield, Russia. In situ generation of fermentation products other than biomass, biopolymers and biosurfactants, for example, gases, alcohols, solvents, or organic acids can also contribute to MEOR.1,5,16

C7H8 + 7.2NO3− + 7.2H+ → 7CO2 + 3.6N2 + 7.6H 2O (2)

when the volumes of the oil and aqueous phases are the same (i.e., 0.5 PV). Reduction of 80 mM nitrate would give 975 and 36 mL of gaseous N2 per liter of aqueous phase at low (1 atm) and high (27 atm) pressure, respectively. Under these conditions bioreactors with high toluene and nitrate gave 16.7 ± 3.9% and 12.3 ± 2.2% more ROIP under low and high pressure conditions, respectively, than bioreactors without added toluene and nitrate (Tables 1 and 2). Likewise, lowpressure bioreactors injected sequentially with high toluene followed by high nitrate gave 17.6 ± 7.0% of additional ROIP (Table S4). The possibility of sequential injection of electron donor and acceptor is a strong point of HN-MEOR. In contrast, when using an aqueous electron donor such as molasses, the mixture of nitrate, molasses, nutrients and F

DOI: 10.1021/acs.est.5b03879 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology These products can swell oil and/or decrease its viscosity, facilitating its displacement.4,16,17 We observed that incubation of bioreactors containing high oil and nitrate gave increases in ΔP of 30 mbar (Figures 3 and S4). This is small, when considering that a ΔP of 3000−4000 mbar is needed to move the viscous (3400 cP), heavy oil phase through the bioreactor, but it is high when considering that a ΔP of only 0.3−0.6 mbar is needed to move the nonviscous aqueous phase (Figure S7). Increased oil production by HNMEOR was associated with increased emulsification (Figure S8), which could be catalyzed by biosurfactant production by the dominant Thauera species (Table S6: 95%) or their attachment to oil needed to metabolize the oil-bound toluene.22 Thauera has also been shown to produce polyhydroxyalkanoate and exopolysaccharide biopolymers,23 which could contribute to MEOR. Hence, the additional oil production observed in HN-MEOR was likely caused by a combination of contributing mechanisms. Increased oil production through sequential injection of light hydrocarbon and nitrate to activate hNRB is a novel MEOR technology. MEOR by injection of oxygen or nitrate with or without the injection of inorganic nutrients using resident hydrocarbon as electron donor is well-known.3,4,6,16,24,25 However, this is technology has not been combined with the injection of specific light hydrocarbons, either in pure form or in mixtures to improve activity of resident nitrate- or oxygen− reducing bacteria downhole. For production of heavy oil HNMEOR is critically dependent on enriching heavy oil in specific substrates, which are limiting. Field applications could, therefore, involve prolonged injection of a water-dissolved low molecular weight hydrocarbon such as toluene followed by injection of a limited volume of a high concentration of nitrate and other needed nutrients (e.g., phosphate, ammonium). Subsequent reduction of nitrate likely requires a period of shutin, as in the experiments presented here. The economics of HN-MEOR can be estimated by considering an oil-bearing formation with a pore volume of 20 000 m3, which has 10 000 m3 of residual heavy oil and 10 000 m3 of water. Amending 10 000 m3 of injection water with 300 ppm of toluene requires 3460 L of toluene at a cost of $3460. Subsequent injection of the equivalent of 80 mM nitrate will require 800 000 mol of nitrate or 400 000 mol (66 tonnes) of calcium nitrate at a cost of $300/ton, that is, a total cost of $20,000. Assuming half the additional oil production as obtained in lab studies (i.e., 5% of ROIP) would yield 500 m3 (3144 bbls), currently valued at $157,233, indicating that HN-MEOR has the potential to be profitable.

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summarize oil and water production in low-pressure bioreactors; Table S5 indicates oil production in high pressure bioreactors; Table S6 summarizes community analyses derived from pyrosequencing data (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 403-220-6388; fax: 403-289-9311; e-mail: voordouw@ ucalgary.ca. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an NSERC Industrial Research Chair Award to GV, which was also supported by BP America Production Co., Baker Hughes Canada, Computer Modeling Group Limited, ConocoPhillips Company, Dow Microbial Control, Enbridge, Enerplus Corporation, Intertek, Oil Search (PNG) Limited, Shell Global Solutions International, Suncor Energy Inc., and Yara Norge AS, as well as by Alberta Innovates-Energy and Environment Solutions. We thank Dr. Rhonda Clark for administrative support and Drs. Yuriy Kryachko and Akhil Agrawal for starting this project.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b03879. Appendix 1 gives details of procedures for high-pressure bioreactor packing and operation and for community analysis by pyrosequencing. Figures S1, S2, S4, S5, S7, and S8 show the set up, oil production, toluene oxidation, nitrate reduction and the relation between water flow rate and pressure gradient in low-pressure bioreactors. Figures S3 and S8 show the set up and oil emulsification activity in high-pressure bioreactors. Table S1 indicates the composition of CSBK medium; Table S2 identifies the enrichment cultures used; Tables S3 and S4 G

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