Investigation of a hydrocarbon-degrading strain, Rhodococcus ruber Z25, for the potential of microbial enhanced oil recovery

Journal of Petroleum Science and Engineering 81 (2012) 49–56

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Investigation of a hydrocarbon-degrading strain, Rhodococcus ruber Z25, for the potential of microbial enhanced oil recovery Chenggang Zheng a,⁎, Li Yu b, Lixin Huang b, Jianlong Xiu b, Zhiyong Huang c a b c

Petroleum Exploration & Production Research Institute, SINOPEC, Beijing 100083, China Institute of Porous Flow & Fluid Mechanics, Research Institute of Petroleum Exploitation and Development (Langfang), China National Petroleum Corporation, Langfang 065007, China Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

a r t i c l e

i n f o

Article history: Received 8 November 2010 Accepted 16 December 2011 Available online 24 December 2011 Keywords: microbial enhanced oil recovery (MEOR) Rhodococcus ruber hydrocarbon mechanism phase behavior waterflooding experiment

a b s t r a c t A hydrocarbon-degrading strain, Rhodococcus ruber Z25, was isolated from the formation brine in Daqing Oilfield, China. The strain Z25 was able to grow under facultative anaerobic condition and produce biosurfactant while hydrocarbon was used as sole carbon source. The biosurfactant of R. ruber Z25 showed a perfect emulsification activity and was able to lower the interfacial tension to approximately 1.0 mN/m and achieved a CMC value of 57 mg/L. The biodegradation experiments of the crude oil by the strain Z25 under aerobic and anaerobic conditions exhibited positive effects in improving the physical properties of the crude oil, including mobility enhancement, cloud point reduction and wax degradation. Waterflooding experiments were done to investigate the MEOR potential of the strain and varied oil recovery efficiencies from 8.88% to 25.78% were achieved. The main MEOR mechanisms of the strain Z25 included hydrocarbon degradation, improvement of oil mobility, wettability alteration and selective plugging. These results revealed that strain Z25 exhibited a tremendous potential for MEOR application. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Microbial enhanced oil recovery (MEOR) has been recognized as a potentially cost-effective method for enhancing oil recovery. The technology is flexible, relatively inexpensive, and can be applied by independent producers. MEOR formulations can be used in a variety of methods including microbial wax removal, single-well treatment (Bryant and Burchfield, 1989), permeability modification treatment (Gullapalli et al., 1998), and microbial enhanced water flooding process (Bryant and Douglas, 1988). Hydrocarbon degradation is the main mechanism for microbial wax removal (Barker et al., 2003; Becker, 2001). Selected inocula and nutrients were injected together to degrade the paraffin and other hydrocarbons that have accumulated on the production equipment, within the well, or within the reservoir (Etoumi, 2007; Ford et al., 2000). The microbial single-well treatment (also called well stimulation) may be localized to the well-bore region or occur several meters to ten or more meters in the reservoir. The objective of well stimulation technologies is to stimulate the production of large amounts of acids, gases, solvents, biosurfactants and/or emulsifiers in the near well region of the reservoir to improve oil rates. In addition to removing scale, wax, asphaltenes, and other debris, well stimulations may change wettability

⁎ Corresponding author at: NO 31, Xueyuan Road, Haidian District, Beijing, China. Tel.: + 86 13520274877. E-mail address: [email protected] (C. Zheng). 0920-4105/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.petrol.2011.12.019

and flow patterns to allow more oil to flow to the well (Grula et al., 1985). Microbial enhanced water flooding differs from the above methods in that the nutrients with or without inocula are injected into the injectors in order to stimulate microbial activity in a large portion of the reservoir and the oil is recovered in producers (Hitzman, 1983; Lazar, 1991). The goal of microbial enhanced water flooding is to increase the ultimate oil recovery of the whole oil reservoir. This is done by improving the microscopic displacement efficiency through a reduction in the capillary forces that entrapped oil or by improving the volumetric sweep efficiency of the recovery fluid by blocking water channels and high permeability zones to push bypassed oil to production wells. The in situ stimulation of hydrocarbon degrading bacteria by injection of oxygen and inorganic nutrients has long been studied to recover additional oil (Andreevskii, 1961; Nazina et al., 2007b). The method has been widely used in Russia and China that very strong evidence links microbial activity with oil recovery (Belyaev et al., 2004; Ivanov et al., 1993; Nazina et al., 2007a). In this approach, the stimulation of aerobic hydrocarbon metabolism in the vicinity of the injection well results in the production of acetate, other organic acids, alcohols and biosurfactants. High concentrations of aerobic hydrocarbon degraders are also detected in fluids close to the injection well that are able to produce biosurfactant while growing on hydrocarbon. A hydrocarbon-degrading strain, Rhodococcus ruber Z25, was isolated from the formation brine in Daqing Oilfield, China (Zheng et al., 2009). The strain exhibits tremendous potential for MEOR application, including hydrocarbon mechanism, facultative anaerobic

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respiration and biosurfactant producing ability. Therefore, the mechanisms for MEOR of the strain Z25 were investigated and the treatment strategies were also discussed in the present work. 2. Materials and methods

Table 2 The physical and chemical parameters of MEOR potential reservoir in Daqing Oilfield. Reservoir parameters Depth, m Pressure, MPa

2.1. Chemicals, media and growth conditions Luria-Bertani (LB) medium was used for preparing the inoculum of the strain. The early log phase microbial culture broth of the bacteria prepared in LB medium was transferred to mineral salts medium (MS medium) at the concentration of 2.0% as the inoculum. The compositions of LB medium, MS medium, the trace elements solution and the vitamin solution were listed in Table 1 (Bao et al., 2009). Liquid paraffin (or crude oil) was supplied as carbon source and added at a concentration of 5.0% (v/v). In anaerobic cultivation, 8.0 g/L of NH4NO3 was added to the MS medium instead of 5.4 g/L NH4Cl to provide the nitrate as alternative electron acceptor for facultative anaerobic respiration. Anaerobic cultivation was conducted as follows (Hao et al., 2008): the water for media preparation was boiled for 20 min in order to dispel all the dissolved oxygen prior to use. L-Cysteine and resazurin as oxygen indicators were added to the medium to final concentrations of 500 mg/L and 100 mg/L, respectively. Pure nitrogen gas was poured into the anaerobic culture bottles until the oxygen indicators in the medium became achromatic. The anaerobic culture bottles were then sealed with rubber caps, sterilized at 121 °C for 20 min before use. The medium was then inoculated via injection. Aerobic and anaerobic cultivations were carried out at 37 °C on a rotary shaker at 150 rpm for 7 days. The formation brine and crude oil were collected from the oil production station of Unit ZX-201 in Daqing Oilfield, China, and were employed for bacterial isolation, oil biodegradation and water flooding experiment. The physical and chemical parameters of MEOR potential reservoir in Daqing Oilfield were listed in Table 2. All the other chemicals in the study were of analytical grade. 2.2. Microorganism Several hydrocarbon-degrading strains were isolated from the production brine of Daqing Oilfield. The strain Z25 was able to degrade hydrocarbon under facultative anaerobic condition and selected for further study. The strain Z25 was identified as R. ruber by 16S rDNA sequencing. 2.3. Biomass and biosurfactant production Members of the genus Rhodococcus are known to produce surfaceactive trehalose-lipids associated with cell growth (Philp et al., 2002). In the present work, the biosurfactant production of R. ruber Z25 was determined via emulsification activity test (data not shown). Liquid

Temperature, °C Pay thickness, m

Formation brine parameters

800–1000 pH 8.3–11.3 Salinity, g/L 35–45 Water type

8.0–8.5 6300– 7000 NaHCO3

45.1–73.4

b0.3

Oxygen, mg/L

Crude oil parameters Density, g/cm3 0.86–0.89 Viscosity (in situ), 9.3–15.5 mPa s Natural gas/crude 38–50 oil ratio

paraffin was supplied as carbon source for biomass and biosurfactant production and the culture was centrifuged at 12,000 rpm and 4 °C for 30 min. The biomass was collected and washed twice with methyl tert-butyl ether (MTBE) to remove residual carbon source and biosurfactant. Then the biomass was dried in an oven at 110 °C to constant weight and the biomass was calculated. The hydrophobic layer located at the surface of the culture was extracted using methyl tert-butyl ether (MTBE) method (Kuyukina et al., 2001). The solvent layer was separated from the aqueous phase and combined with the cell-washing MTBE for biosurfactant isolation. The solvent was removed by rotary evaporation at 50 °C under reduced pressure. The extract was then thoroughly washed thrice with petroleum ether to remove residual carbon source to obtain crude biosurfactant. The crude biosurfactant was then freezedried and stored under nitrogen.

2.4. SFT/IFT and contact angle measurements Surface tension (SFT) and interfacial tension (IFT) were measured by ring Du Nuoy method using a Kruss K100 Tensionmeter (Hamberg, Germany) at room temperature. As crude biosurfactant was sparingly soluble in water, its SFT and IFT measurements were performed immediately after emulsification by ultrasonic treatment in the formation brine (20 kHz, 1 min). Interfacial tension was determined against n-hexadecane. Critical micelle concentration (CMC) was calculated as the lowest concentration at which the SFT and IFT values were minimal. The contact angle was measured using a Thermo Cahn Automated dynamic contact angle (DCA) analyzer (Cahn, USA) (Serro et al., 1997). The analyzer was operated by holding a quartz plate in a fixed vertical position, attaching it to a microbalance and moving a probe liquid (the formation brine) at constant rate up and down past the plate (Fig. 1). A unique contact angle hysteresis curve was produced by the microbalance as it measured the force exerted by the moving contact angle in advancing and receding directions. The dynamic contact angle was then calculated from the modified Young's Equation (Wilhelmy Equation). The DCA measurements were conducted in duplicate.

Table 1 The compositions of media in the present study. Luria-Bertani (LB) medium

Mineral salts (MS) medium

Peptone, g/L Yeast extract, g/L NaCl, g/L pH

KH2PO4, g/L K2HPO4, g/L NH4Cl, g/L MgSO4·7H2O, g/L CaCl2·2H2O, g/L FeSO4, g/L Trace elements solution, ml/L Vitamin solution, ml/L pH

10.0 5.0 10.0 7.0–7.2

Trace elements solution 1.0 1.0 5.4 0. 0.01 0.01 1.0 1.0 7.5

H3BO3, mg/L ZnSO4·H2O, mg/L MnCl2·4H2O, mg/L CuSO4·5H2O, mg/L NiCl2·H2O, mg/L Na2MoO4·H2O, mg/L

Vitamin solution 280.0 178.0 8.0 8.0 80.0 50.0

Bioton, mg/L Thiamine HCl, mg/L Thioctic acid, mg/L Nicotinic acid, mg/L Riboflavin, mg/L Pyripoxin HCl, mg/L D-Calcium Pantothenate, mg/L Folic acid, mg/L Cyanocobalamin, mg/L P-aminobenzic acid, mg/L

2.0 5.0 5.0 5.0 5.0 10.0 5.0 2.0 0.1 5.0

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Fig. 1. Contact angle measurement.

2.5. Characterization of the crude oil properties

2.7. Water flooding experiment

After the cultivation, the cultures were centrifuged at 8000 rpm and 4 °C for 20 min in order to separate and obtain the crude for the following measurements. The negative control was prepared using the same method except uninoculated. The viscosity measurements of the crude oil and the emulsions (in Section 2.6) were carried out using a Brookfield DV-II programmable viscometer (Brookfield, USA), fitted with thermos system for the measurements at 50 °C. The measurements were taken when the samples were at equilibrium. That was at point when increased in the shear rate (related to spindle rotation) resulted in no or little change in the viscosity. In other words, the samples exhibited signs of a Newtonian fluid. The cloud point of the crude oil was measured using a SYD-510 Cloud Point analyzer, (Jichang Co., Shanghai, China). The oil samples for cloud point test were maintained at desired temperature above the cloud point in a piston transfer cylinder. The ice-water bath was programmed to decrease the temperature at the predetermined rate and the samples were examined periodically. The temperature at which a cloud was first observed at the bottom of the cylinder was recorded as the cloud point. The wax and asphaltene contents in crude oil were determined according to the standard (SY/T 7550-2004) in China. The crude oil for wax content measurement was prepared in an alumina PLOT column. The wax was extracted by methylbenzene and dried at 110 °C under reduced pressure. The asphaltene content was determined by n-heptane extraction and precipitation. The saturated hydrocarbon composition of the crude oil was determined by the gas chromatography (GC) method. The oil samples were first extracted by chloroform and then measured using HP 6890 GC, equipped with a PONA quartz capillary column (30 m × 0.2 mm × 0.2 μm). Split injections were conducted using nitrogen as carrier gas. The column temperature increased from 50 °C to 310 °C at a rate of 8 °C/min. An interface temperature of 310 °C and an ion source temperature of 320 °C were utilized.

The potentials of microbial enhanced oil recovery by the strain R. ruber Z25 and its metabolite were evaluated using the sand package technology (Suthar and K., H., 2008). A column of 25.0 × 3.0 cm dimensions with a sieve and cap fixed at the bottom was packed with acid washed quartz sand. Each sand package core was first evacuated and the afterwards flushed with N2 for absolute gaseous permeability determination. After that, the cores were evacuated again and vacuum-saturated with sterile formation brine for porosity and absolute aqueous permeability determination. Sand package cores were then saturated with crude oil until residual water saturation was reached. The original oil in place (OOIP) equaled the volume of brine displaced by oil flooding. All oil-saturated cores were subsequently waterflooded with sterile formation brine until no more oil was observed in the effluent, i.e., until the residual oil saturation was reached. The amount of crude oil retained in the sand package core was determined volumetrically. In order to evaluate the potential of strain Z25 for MEOR application, a series of waterflooding experiments was performed under the typical strata conditions in Daqing Oilfield of China, i.e., under a pressure of 10.0 MPa at 37 °C. The cores for each in situ MEOR experiment were inoculated and cultured anaerobically. For the ex situ MEOR experiments, crude biosurfactant suspension was injected into the cores to determine whether they affected the oil mobilization process. After the shut-in period of 20 days, the cores were flooded again with the same sterile formation brine. The flow rate was approximately 0.50 ml/min and the injection pressure was monitored throughout the experiment.

2.6. Emulsification activity and phase behavior The phase behavior of biosurfactant/oil/brine system was studied by mixing the formation brine, biosurfactant and crude oil (liquid paraffin) at different oil–water ratios and different biosurfactant concentrations. The systems were mixed using a vortex mixer and the viscosity of the emulsion was immediately measured as described in Section 2.5. The type of emulsion was determined by using methyl orange and Sudan red III (Tian et al., 1998). The values of the volume percentage of water phase, oil phase and emulsion phases were monitored after 24 h in order to insure that the equilibrium of the emulsion was reached (Abdulrazag and Al-Khanbashi, 2000).

3. Results 3.1. Biomass and biosurfactant production Aerobic and anaerobic cultivations were conducted in order to determine the cell growth and biosurfactant production on hydrocarbon (liquid paraffin). Under aerobic condition, the biomass and biosurfactant production of the strain Z25 were 1.46 g/L and 12.95 g/L, respectively. The strain Z25 was not able to grow without the nitrate supply under the anaerobic condition, indicating that nitrate worked as an available electron acceptor under anaerobic condition and nitrate reduction activity played a crucial role in hydrocarbon degradation and microbial growth (Baek et al., 2003; Cunningham et al., 2000). The biomass and biosurfactant production (0.11 g/L and 0.53 g/L, respectively) achieved on mineral salts medium under anaerobic condition were much lower than those obtained under aerobic condition. Though the microbial growth and metabolic activities were partially inhibited under the anaerobic condition, the metabolic activity provided an alternative way of stimulating hydrocarbon-degrading bacteria in the oil reservoir without oxygen introduction.

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Surface tension, mN/m

a 80

Table 4 Alteration of the crude oil quality by aerobic and anaerobic treatments.

70 60

Viscosity (50 °C), mPa s Cloud point, °C Wax content, % Asphaltenes content, % Alkane compositions Max peak C22 −/C23 + C6–C16, % C17–C34, % C35–C45, %

50 40 30 20

0

50

100

150

200

250

300

350

400

Negative control

Aerobic cultivation

Anaerobic cultivation

20.8 29.5 14.46 11.38 C23 0.71 26.79 70.05 3.16

16.4 26 11.80 12.14 C23 0.61 17.13 77.38 5.49

18.2 27 13.76 11.93 C23 0.75 20.92 76.91 2.17

450

Biosurfactant concentration, mg/L wetting. In the present work, the quartz plates were first soaked in a hydrophobic substance (liquid paraffin or crude oil) at 37 °C for 3 days to obtain a hydrophobic wetting surface. Then the plates were treated again with microbial culture broth (sterile or not) and the contact angles before and after the treatment were monitored (Table 3). All the quartz plates exhibited positive response due to the microbial treatment, changing the hydrophobic wetting surfaces to hydrophilic ones or at least less hydrophobic ones. The quartz plates treated by wax exhibited stronger hydrophobic wetting surfaces than those treated by crude oil. However, after the microbial culture broth treatment, the plates showed greater hydrophilic wetting surfaces than those treated with sterile culture broth.

Interfacial tension, mN/m

b 50 40 30 20 10 0 0 -10

25

50

75

100

125

150

175

200

225

3.3. Biodegradation and physical properties alteration of the crude oil

Biosurfactant concentration, mg/L

Fig. 2. Surface tension and interfacial tension of the crude biosurfactant of Rhodococcus ruber Z25.

The result above also suggested that the biosurfactant production by R. ruber Z25 was primarily cell-growth associated when the strain grew on hydrocarbon, similar to that reported by other investigators (Rodrigues et al., 2006). An oil/water emulsion was formed in the culture and the emulsion could keep stable for more than three months, suggesting that the biosurfactant was extremely effective in emulsification ability. 3.2. SFT/IFT and contact angle measurements The crude biosurfactant was separated by MTBE method and appeared to be a reddish brown powder. After dissolved in the filtered formation brine, the SFT and IFT values were determined. The crude biosurfactant solution could decrease SFT from 68.574 mN/m to 29.540 mN/m and achieved a CMCSFT value of 133 mg/L (Fig. 2a). Interfacial tension against n-hexadecane decreased from 43.621 mN/m to approximately 1.0 mN/m and a CMCIFT value of 57 mg/L was monitored (Fig. 2b). Contact angle measurements obtained by Thermo Cahn DCA analyzer range from 0° to 180°. Hydrophilic wetting was an effect commonly characterized by a 0°, or close to 0° of contact angle that allowed an aqueous liquid to easily spread over the quartz surface while a 180°, or close to 180° of contact angle suggested hydrophobic

The biodegradation experiments of the crude oil were conducted under aerobic and anaerobic conditions. Both the aerobic and anaerobic treatments exhibited positive effects on improving the physical properties of the crude oil. As shown in Table 4, the viscosity of the crude oil decreased from 23.8 mPa s to 16.4 mPa s and 18.2 mPa s for aerobic and anaerobic treatments, respectively. Meanwhile, the cloud points of the crude oil after aerobic and anaerobic treatments reduced from 29.5 °C to 26 °C and 27 °C, respectively. These results were inconsistent with the changes of wax contents of the crude oil, which dropped from 14.46% to 11.80% and 13.76% for aerobic and anaerobic treatments, respectively. However, the asphaltene contents of the treated crude oil were found to increase slightly compared with the negative control. A vast array of microbial species have been found to be able to degrade low- (three rings or fewer) and highmolecular-weight (four rings) polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, acenaphthene, anthracene, fluoranthene, pyrene, and chryseneas as sole carbon and energy sources. However, no strains have yet been found to utilize PAHs with more than four rings as a sole carbon and energy source. Therefore, the asphaltenes might not be utilized by the strain Z25 as the carbon source in the oil reservoirs or the present experiment and the increase of asphaltenes content mainly resulted from the consumption of the saturated hydrocarbons including the wax. In this study, the compositions of the saturated hydrocarbons were analyzed by GC method. Long-chain alkanes could not be converted to short-chain ones that there are no microorganisms known to catalyze such a reaction. In fact, a number of literatures have

Table 3 Contact angle measurements of the quartz plates. Quartz plates

Test I Test II

Sterile culture broth

After After After After

treatment of wax microbial treatment treatment of crude oil microbial treatment

Microbial culture broth

Advancing angle, °

Receding angle, °

Advancing angle, °

Receding angle, °

155.58 ± 11.49 90.05 ± 8.35 136.43 ± 13.33 82.40 ± 15.41

116.86 ± 6.67 64.33 ± 12.20 93.67 ± 13.42 74.41 ± 13.94

146.40 ± 9.84 53.24 ± 7.13 133.39 ± 11.16 81.29 ± 7.71

128.27 ± 10.70 27.51 ± 20.62 77.35 ± 7.50 48.90 ± 19.16

C. Zheng et al. / Journal of Petroleum Science and Engineering 81 (2012) 49–56

reported changes in the alkane compositions of the crude oil due to the microbial treatment (He et al., 2003; Wankui et al., 2006). Some strains were reported to utilize a wide range of alkanes (C12–C36) or a narrow range of substrates (Koma et al., 2001). A relative wide range of alkanes (C6–C45) could be utilized by the strain Z25 according to GC analysis (Fig. 3). However, the preferences of alkane utilization by the strain R. ruber Z25 under aerobic and anaerobic conditions were quite different. The max peak of alkanes of the crude oil appeared at C23. Under the aerobic condition, the ratio of C22 −/C23 + decreased from 0.71 to 0.61, suggesting that the short-chain alkanes were utilized preferentially. C6–C16 components were of the most preferential utilization, reducing from 26.79% to 17.13%. Meanwhile, under the anaerobic condition, long-chain alkanes were utilized preferentially. The ratio of C22 −/C23 + increased from 0.71 to 0.75 and C35–C45 components decreased from 3.16% to 2.17%. The mechanisms underlying hydrocarbon degradation under anaerobic condition were still unclear and one possible reason was that it was economics for cells to uptake long-chain alkanes in hydrocarbon transportation under anaerobic conditions (Liu et al., 2006).

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3.4. Phase behavior study Biosurfactant producers isolated from a number of oil reservoirs are effective in reducing SFT/IFT values and mobilizing residual oil in a variety of laboratory conditions (Bodour and Miller-Maier, 2002; Youssef et al., 2005). However, very limited laboratory work has focused on phase behavior of the biosurfactant. In the present work, an investigation on phase behavior of the biosurfactant was conducted. Fig. 4 exhibited the values of phase volume percentage of biosurfactant/oil/brine systems at different oil–water ratios and biosurfactant concentrations. The emulsion types of the biosurfactant/oil/brine systems were found to change with biosurfactant concentrations and oil–water ratios. With the increase of biosurfactant concentration from 0.5 g/L to 2.0 g/L, the water/oil emulsion had to be converted to oil/water emulsion at oil–water ratios of 60:40 and 70:30. Comparing the ultimate water phase percentage with the initial water fraction in the biosurfactant/oil/brine systems, very small fraction of water goes to the oil/water emulsion while relative large water fraction is into the water/oil emulsion. Another significant

Fig. 3. GC analysis of n-alkane compositions of crude oil after aerobic and anaerobic treatments of the strain Rhodococcus ruber Z25. (a) Negative control; (b) aerobic biodegradation; (c) anaerobic biodegradation.

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0.5g/L of crude biosurfactant Phase percentage, %

100% 80%

oil phase

60%

water/oil emulsion oil/water emulsion

40%

water phase

their metabolites with oil recovery enhancements of 8.88% OOIP and 9.13% OOIP. In ex situ MEOR experiments, additional oil recoveries with 1.0 g/L and 2.0 g/L biosurfactant solution treatments were 16.51% OOIP and 25.78% OOIP, respectively, suggesting a potential of ex situ MEOR application. The resistance factor (RF) was employed to investigate the injecting pressure variation and the RF values varying from 1.55 to 5.96 were monitored in the MEOR experiments, presenting a corresponding increase with the oil recovery enhancement.

20% RF¼

0%

Equilibrium pressure after MEOR Equilibrium pressure before MEOR

ð1Þ

Water-oil ratio 4. Discussion

1.0g/L of crude biosurfactant Phase percentage, %

100% 80%

oil phase

60%

water/oil emulsion oil/water emulsion

40%

water phase

20% 0%

Water-oil ratio 2.0g/L of crude biosurfactant Phase percentage, %

100% 80%

oil phase

60%

water/oil emulsion oil/water emulsion

40%

water phase

20% 0%

Water-oil ratio Fig. 4. Phase behavior at different water–oil ratios and biosurfactant concentrations.

observation was that the viscosity of the emulsions correlated significantly with the emulsion type. The oil/water emulsion exhibited a perfect fluidity, with a viscosity of approximately 1.0 mPa s at 50 °C, while the water/oil emulsion showed poor fluidity with a viscosity of more than 2000 mPa s. 3.5. Water flooding experiment In the water flooding experiment, the oil recovery enhancements resulting from metabolic activities of R. ruber Z25 (in situ MEOR) and crude biosurfactant (ex situ MEOR) were determined. The water flooding only recovered approximately 50% of crude oil until no more oil was produced. After the shut-in period, the cores were water flooded again with the formation brine to investigate the ultimate oil recovery and MEOR efficiency. A negative control was employed for there would be a redistribution of the residual oil in the porous media during the shut-in period and an enhancement of oil recovery (4.21% OOIP). As shown in Table 5, the residual oil in the cores was found to be mobilized again by the cells growth and

In the present work, a hydrocarbon-degrading strain R. ruber Z25 was isolated from the formation brine in Daqing Oilfield, China. The strain Z25 was able to grow under facultative anaerobic condition and produce biosurfactant on hydrocarbon. Therefore, the strain was selected for investigating its potential application in microbial enhanced oil recovery in the oil reservoir. The removal of wax from the well and production equipment could reduce operating costs and improve the flow of oil into the well by altering drainage patterns and/or fluid saturations near the well. Chemical methods include the use of solvents, surfactants, dispersants, and wax crystal inhibitors that may cause serious formation damage and environmental problems (Singh et al., 2007). Thermal methods include the treatment of wells with hot fluids, usually hydrocarbons or water, to remove deposits, which result in huge energy consumption (Etoumi, 2007). Stimulation of in situ hydrocarbon metabolism is the most common microbial approach to treat wax deposition problems, which can reduce the cost and environmental risk (Spormann and Widdel, 2001; Van Hamme et al., 2003). The strain Z25 exhibited the desirable properties for applications in wax removal, including alkane degradation, cloud point reduction, and fluidity improvement. In the contact angle experiment, the quartz plates treated by the wax substance showed an obvious layer of wax deposit on their surfaces. Sterile microbial broth treatment could only alter the wettability to some degree that the plates still exhibited a strong hydrophobic surface after the treatment, indicating that single biosurfactant treatment was not efficient enough to remove the wax deposit. On the contrary, after the microbial treatment, the quartz plates' surfaces converted to hydrophilic wettability (Table 3). The cells growth on hydrocarbon did not only mediate the mobility of the crude oil (biosurfactant production) and the cloud point reduction (hydrocarbon degradation), but also destroyed the structure of wax deposit and dispersed the debris due to the adherence of hydrophobic bacteria at the oil–water interface (Dorobantu et al., 2004), indicating that the in situ hydrocarbon mechanism must be involved in the wax removal process. Well stimulation technologies are not technically EOR processes, however, these processes could greatly extend the economic life of the production well, either by reducing operating costs or increasing daily revenue. Therefore, more oil could be recovered from the reservoir at a relative low price. Numerous field trials have reported in situ hydrocarbon degradation and biosurfactant production mechanisms or ex situ biosurfactant injection for well stimulation. The production wells always fail to work efficiently for the sake of scale and/or wax deposit, asphaltenes precipitation and debris formation. Alterations of the physical properties of crude oil such as the viscosity, fluidity and mobility may result from the production of biosurfactants or other surface active compounds in terms of low viscosity oil/water emulsion and plug removal (Etoumi, 2007; Rosenberg and Ron, 1999). Moreover, the cells growth could also change the surface wettability of the porous media (as shown in Table 3) and hence alter the

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Table 5 In situ and ex situ MEOR water flooding experiments. Core 1a

Core 2b

Core 3b

Core 4c

Core 5c

Porosity (φ), % Pore volume (PV), ml Absolute gaseous permeability (Kg), 10− 3 μm2 Absolute gaseous permeability (Kw), 10− 3 μm2 Original oil in place (OOIP), %PV Water flooding oil recovery, %OOIP Equilibrium pressure before MEOR, MPa MEOR treatment

34.54 33.90 315 240 80.53 50.28 0.21 0.5 PV formation brine

Total oil recovery, %OOIP MEOR efficiency (EMEOR), %OOIP Equilibrium pressure after MEOR, MPa Resistance factor, RF

54.49 4.21 0.22 1.05

34.05 33.60 323 257 79.62 51.96 0.21 0.5 PV MS medium with 5% inoculum 60.84 8.88 0.36 1.76

33.27 32.60 305 222 85.42 50.27 022 0.5 PV MS medium with 5% inoculum 59.40 9.13 0.34 1.55

32.52 31.91 346 296 81.25 52.02 0.24 0.5 PV of 1.0 g/L biosurfactant solution 68.53 16.51 0.85 3.54

33.53 33.90 343 293 84.31 55.73 0.23 0.5 PV of 2.0 g/L biosurfactant solution 81.51 25.78 1.37 5.96

a b c

Negative control. In situ MEOR. Ex situ MEOR.

flow patterns in the well-bore region by adjusting the relative permeability curve of oil–water phase (Kowalewski et al., 2006). Microbial enhanced water flooding has significant potential for increasing production from aging oil fields that are currently undergoing water flooding. The incremental cost for injecting microbes and nutrient is relatively small in an existing water flood system, which may make this recovery method applicable at low oil prices when more expensive methods are not economically feasible. At the end of water flooding, the high capillary pressure traps crude oil in small pores within the rock matrix that in order to recover this trapped oil, the interfacial tension between oil and aqueous phase should lower to 10 − 3 mN/m or even 10 − 4 mN/m (McInerney et al., 2005). Up to now, none of biosurfactant has been proved efficient to lower IFT value to such degree. The crude biosurfactant in this study lowered IFT merely to 1.0 mN/m, on the contrary, significant oil recovery enhancements were monitored (Abdulrazag et al., 1999; Nourani et al., 2007), suggesting that IFT was not the exclusive factor in evaluating the MEOR efficiency. R. ruber Z25 was able to grow on hydrocarbon and alter the physical properties of the crude oil. However, the in situ growth of the hydrocarbon-degraders was severely restricted by the electron acceptor supply, and in the pilot trials in China, very small MEOR plug for treatment (less than 0.001 PV of the oil reservoir) was employed. Therefore, the hydrocarbon-degrading activities could only be achieved in less than tens of meter zones near the injection well-bore region. In the present work, a facultative hydrocarbon-degrader was employed and this can provide a new measure of supplying electron acceptor and insuring the active hydrocarbon degradation. In order to characterize the MEOR process of hydrocarbon mechanism, the phase behavior of the biosurfactant was also investigated and the viscosities of different types of emulsion were monitored. According to the results of phase behavior, it can be inferred that in the near well-bore region of the injector, where the saturation of residual oil is relatively low, the biosurfactant in the brine (supplied by injection or in situ production) could result in the oil/water emulsion formation. Under the given condition, the trapped oil in these areas will be stimulated again during the shut-in period and flushed later with the recovery fluids toward the producer. During the transport process of oil/water emulsion in the porous media, the physical and chemical conditions will change greatly. First, the biosurfactant concentration continues to decrease due to dilution and pervasion of the emulsion. Second, saturations of the oil/water phases change a lot that water rich zone is found at the injectors' region while oil rich zone is often monitored in deep part of oil reservoir and producers' region. Moreover, the emulsions may also be trapped in the porous throat, congregating with each other, and/or they may converge with the adjacent oil droplet on the surface of rock matrix. As

shown in phase behavior study, physical and chemical changes will definitely affect the emulsion and type transformation of the emulsion would be likely to happen. It can be inferred that as dilution of biosurfactant in the emulsion droplet and/or increase of crude oil saturation, some of the oil/water emulsions could convert to water/oil ones. The physical properties of the emulsion will also change with the emulsion type transformation and the newly-formed viscous emulsion can further block the porous media, which in turn influence the flow pattern by selectively plugging the existing water channels in the oil reservoir. For most of the emulsions transport along the high permeability (water rich) zones, an enhancement of flowing resistance in these areas will come into being and hence track the post recovery fluids to the bypass zones of higher residual oil saturation. Besides, the increase of pressure gradient in the porous media will result in enhancements of capillary number and water flooding efficiency as well. As a result, the total oil recovery will be enhanced by improving the microscopic displacement efficiency and the volumetric sweep efficiency. This deduction would best explain the exact MEOR process in the present work and at least in some of the field trials. As shown in Table 5, the RF values increased significantly in the experimental cores varying from 1.55 to 5.96 comparing with 1.05 in negative control and the MEOR efficiencies were found to increase correlatively with the RF values, suggesting that the discussed mechanisms could probably work in the MEOR application. Although many other factors in the oil reservoir would affect the MEOR efficiency and the application feasibility of this method still requires further research, this study presents a novel view of explaining and characterizing the mechanisms of microbial enhanced oil recovery. The results obtained from the present work also demonstrated that the strain Z25 had a great potential in MEOR application. Acknowledgment This work was supported by State Key Development Program of Basic Research of China (No. 2005cb221308) and National Natural Science Foundation of China (No. 51104106/E0403). References Abdulrazag, Y., Al-Khanbashi, A., 2000. Microbial phase behavior laboratory studies. International Petroleum Exhibition and Conference. Society of Petroleum Engineering, Abu Dhabi, U.A.E. Abdulrazag, Y., Almehaideb, R.A., Chaalal, O., 1999. Project of increasing oil recovery from UAE reservoirs using bacteria flooding. Proceedings of the SPE Annual Technical Conference. Society of Petroleum Engineers, Richardson, Texas. Andreevskii, I.L., 1961. The influence of the microflora of the third stratum of the Yaregskoe oil field on the composition and properties of oil. Trudy Inst. Mikrobiol. 9, 75–81.

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