Selection of Bacteria with Favorable Transport Properties

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, p /83/ $02.00/0 Copyright C) 1983, American Society for Microbiology Vol. 46, No. 5 Selection of Bacteria with Favorable Transport Properties Through Porous Rock for the Application of Microbial- Enhanced Oil Recovery LONG-KUAN JANG, PHILIP W. CHANG, JOHN E. FINDLEY, AND TEH FU YEN* School of Engineering, University of Southern California, University Park, Los Angeles, California Received 29 April 1983/Accepted 2 September 1983 This paper presents a bench-scale study on the transport in highly permeable porous rock of three bacterial species-bacillus subtilis, Pseudomonas putida, and Clostridium acetobutylicum-potentially applicable in microbial-enhanced oil recovery processes. The transport of cells during the injection of bacterial suspension and nutrient medium was simulated by a deep bed filtration model. Deep bed filtration coefficients and the maximum capacity of cells in porous rock were measured. Low to intermediate (_106/ml) injection concentrations of cellular suspensions are recommended because plugging of inlet surface is less likely to occur. In addition to their resistance to adverse environments, spores of clostridia are strongly recommended for use in microbial-enhanced oil recovery processes since they are easiest among the species tested to push through porous rock. After injection, further transport of bacteria during incubation can occur by growth and mobility through the stagnant nutrient medium which fills the porous rock. We have developed an apparatus to study the migration of bacteria through a Berea sandstone core containing nutrient medium. The utilization of microbiological technology to recover residual oil left after secondary waterflooding of the oil reservoir has been demonstrated in Eastern Europe (1, 5). Recently, microbial-enhanced oil recovery (MEOR) has attracted worldwide attention in the petroleum industry. This advanced technology is actually a resurgence which began with the pioneering research of ZoBell in the late 1940s (7, 8). Usually three successive stages are involved in the operation of a typical in situ MEOR process (1, 5, 6). First, potential bacteria or dormant spores are injected along with nutrient into the candidate reservoir, usually one that has been waterflooded. Second, the injection well and the production well are sealed off for a period of a few months. In this period, the injected bacteria multiply and migrate in the reservoir. The nutrient is converted into biogas and other metabolites. Third, at the completion of the in situ incubation, the production well is opened. The pressure derived from the biogas pushes out some of the residual oil. (Sometimes oil is produced from the same injection well, a process usually referred to as "huff-and-puff.") A waterflood follows to displace the oil being released by mobility-modifying metabolites such as organic acids, organic solvents, biosurfactants, and biopolymers. One might conclude from the literature that the success of an MEOR process depends on the selection of the candidate reservoir, the proper choice of potential bacterial species, the viability of bacteria under reservoir conditions, the amount of metabolites generated and their effects on releasing residual oil, the spreading of bacteria and nutrient in the reservoir, and other economic factors. It was generally believed that one major advantage of MEOR processes over other tertiary recovery processes is that active components capable of releasing oil are generated in situ instead of being supplied ex situ. Besides, the nutrients used, such as molasses and low-grade proteins, are generally cheaper than other chemicals used in conventional tertiary recovery. However, microbial treatment of a reservoir did not always yield a significant increase in oil production. It has been established that high porosity and high permeability of the reservoir promises positive results (1). It was also reported that for reservoirs having a permeability higher than 0.6 darcy an area of 60,000 m2 was affected by microbial treatment. (One darcy is ca cm2. Darcy's Law correlates the superficial velocity [U], permeability [k], liquid viscosity [,u], and pressure drop per unit length in porous media [dpidz] as: U = -[kil,][dpidz].) For reservoirs with tight formations having a permeability around 0.1 darcy, the effect was limited to the well bore region (1). One might 1066

2 VOL. 46, 1983 speculate that porous media with higher permeability offer less resistance to the transport of bacteria, and, therefore, a larger fraction of reservoir is influenced by microbial treatment. Thus, in considering the feasibility of MEOR, an exceedingly important parameter is the transport of bacteria within a formation. However, very little quantitative data exist on the relationship between microbial transport in porous media and the efficiency of MEOR. Therefore, among the factors influencing the efficiency of MEOR processes, we initiated a study of bacterial transport in porous media. The transport of bacterial cells (or spores) in the first stage of operation, i.e., the injection of bacterial suspension by pumping, is simulated by use of a deep bed filtration model. Since the sizes of cells are much smaller than the average pore size of a highly permeable porous rock, cells traveling with carrier medium are gradually retained by the porous rock. The probability of retaining a cell as it travels with the suspending media is related to the structure of porous rock, the flow rate, and the flow pattern, as well as the long-range (about 1,000 nm) and the short-range (below 10 nm) colloidal forces that exist between cellular surface and rock. Further complications of concern are the specific cellular attachment mechanisms and the presence of residual oil in the porous media. Sometimes certain bacteria might sense particular chemical attractants (or repellents) such as nutrients adsorbing onto the rock surface and develop a strong directional diffusion toward (or away from) the source of attractants (or repellents), a phenomenon usually called chemotaxis. To obtain sonre quantitative data on the injection of bacterial suspension, a one-dimensional sandstone core with length L was used in this study. Assume that at time t = 0, the injection of bacterial suspension is started. The superficial velocity of injection is U (= volumetric flow rate/cross-sectional area of the core). The space time T at any location Z from the influent surface is defined (2) as: v= t- U (1) At the inlet end, T is equivalent to t. It is convenient to describe the dynamics of injecting bacterial suspension in terms of T. In this manner, the "observer" travels with the carrier medium at the same velocity instead of observing at a fixed point outside the system. It is apparent that the space time T has a fixed value during the period the observer travels in the core. If the probability of retaining a cell as it travels a unit length in the core with carrier medium is a constant, K0, the observer entering MICROBIAL-ENHANCED OIL RECOVERY 1067 at T = t will detect an exponential decay in cellular concentration around him as he travels in the core (2) before the influent surface is saturated: Cz = Ci exp(-koz) (2) where Ci is the influent concentration. As a result of continued deposition of cells onto rock grains occurring before space time T, the observer will also detect an exponential decay in the number of cells deposited per unit of bulk volume of the rock: Uz = KOUC,-r exp(-koz) (3) When the observer leaves the system, the cellular concentration in aqueous phase reduces to CL, and, therefore, the deep filtration coefficient KO can be calculated from equation 2 as: ln (C,/CL) KO = (4) As the deposition of cells continues, at some instant of time tmax the influent surface will be saturated, i.e., the amount of deposition is at its maximum value, Umax: omax = UCiKotmax = UCiKoTmax (5) and the influent surface cannot retain any more cells injected into the system. As the result of the subsequent deposition in the downstream of the influent surface, the "saturation front" will move at a velocity (2) V= U Cj Cmax toward the effluent end. Meanwhile, the effluent concentration increases. Finally, the whole core is saturated, and a breakthrough of cellular concentration in the effluent is observed. Figure 1 demonstrates the distribution of cells in the aqueous phase and on rock grains. For T < TMmax, C versus Z is represented by a single curve (equation 2). The amount of deposition a increases from zero to the curve represented by: cjz = K,UCj Tmax exp(-koz) (7) For T > Tmax, the two curves (equations 2 and 7) shift toward the effluent end at a velocity V. A typical history of effluent bacterial count based on this mechanism is illustrated in Fig. 2. Therefore, if one monitors the variation of effluent bacterial count, the distribution of cells in the core can be fully described by the parameters K0, U, and Tmax, /which can be experimentally obtained. (6)

3 1068 JANG ET AL. Ci L) A inlet EXP(-KoZ) a = KOU C1T EXP(-K%Z) (T r.x) zt/7kix KSzz;1 CZ = Ci inlet FIG. 1. The distribution of bacteria (A) in the aqueous phase and (B) on the rock surface versus space time T (defined by equation 1) according to the deep bed filtration model (2). Symbols: C, aqueous phase concentration; a, amount of deposition; Z, distance from the influent surface. In the second stage of operation in MEOR processes, i.e., the in situ incubation of bacteria and the generation of oil-releasing metabolites, the further transport of bacteria can be achieved through bacterial growth and migration in the porous media. The migration rate is governed by the Brownian motion, the bacterial motility, the growth and death rate of bacteria, the amount of available nutrient, the porous structure of rock, and the adsorption of cells onto the rock surface. In this paper, we present bench-scale experiments showing the relative mobility of bacteria through the stagnant nutrient in an oil-free porous rock. Ci T<?mx 0~~~ amox vmx= = tmoxuci Ko Ctsot-t1mo) K =In [C / U Cj APPL. ENVIRON. MICROBIOL. MATERIALS AND METHODS Porous rock. The Berea sandstone cores with permeability ranging from 0.4 to 0.5 darcy were obtained from Cleveland Quarries Co., Cleveland, Ohio. Another sandstone was obtained from the road-cut of Lake Keystone, Okla. It has a higher permeability of 4 darcy and is called Cleveland sandstone in this work. The core was cut into sections 3 in. (7.62 cm) long and was mounted in a stainless steel core holder. After autoclaving at 125 C for 40 min, the core was saturated with degassed sterile brine by vacuum. A 75% alcohol flood followed by a large amount of sterile brine was injected to sterilize the pump and the pipeline and to flush the core. In one of the experiments, the core was charged with residual oil by injecting 4 pore volumes of crude oil from the Ranger Zone, Long Beach, Calif., into the brine-saturated core. The oil in the core was further displaced by injecting a large amount of sterile brine until the effluent contained very little oil. The oil remaining in the core is called the residual oil in this work. Negatively charged microspheres. In the preliminary stage of this research, negatively charged microspheres were used to simulate the bacterial cells (3). It was found that microspheres were strongly retained by Berea sandstone. In the present work, microspheres were injected into the Berea sandstone core containing residual oil. The main purpose was to investigate the influence of oil on the transport of microspheres. Bacterial species. Pseudomonas putida (ATCC 12633), Bacillus subtilis, and Clostridium acetobutylicum were used. They were cultivated in nutrient broth (0.5% peptone, 0.3% beef extractives) at 32 C. Filtered nitrogen was purged into the cultivating broth of the anaerobe, C. acetobutylicum. B. subtilis and C. acetobutylicum developed spores as the culture aged. These three species were among the bacteria which were found to have potential application in MEOR I pv tmox tsot FIG. 2. A typical curve of effluent bacterial count versus injection time t. Abbreviations: L, length of the sandstone core; U7max, maximum capacity of bacteria per unit bulk volume of rock; U, superficial velocity; K0, filtration coefficient; C, and CL, influent and effluent bacterial concentrations, respectively; pv, pore volume (2). FIG. 3. The schematic diagram of the apparatus for injecting suspension. 1, Oven; 2, core holder; 3, differential pressure transducer; 4, signal demodulator; 5, recorder; 6, valve; 7, high-performance liquid chromatography pump; 8, magnetic stirrer; 9, bacterial suspension; 10, back pressure controller.

4 VOL. 46, 1983 MICROBIAL-ENHANCED OIL RECOVERY 1069 FIG. 4. The apparatus for investigating the bacterial migration through stagnant nutrient broth in the Berea sandstone core. 1, Cotton plug; 2, 250-ml flask; 3, Berea sandstone core; 4, bacterial culture; 5, sterile nutrient broth (turns cloudy after bacteria from the bacterial culture [4] arrive and grow). processes. The metabolites generated during in situ fermentation, i.e., biopolymers (by P. putida), biosurfactants (by B. subtilis), and biogas, solvents, and organic acids (by C. acetobutylicum), can improve the mobility of the oil trapped in the reservoir rock. For this reason, we particularly chose these three species and studied their transport in porous media for this work. Injection of bacterial suspension. The schematic diagram of the apparatus is shown in Fig. 3. The cells were separated from the fermentation broth by centrifugation and were suspended in sterile brine containing 1,000 ppm (1,000,ug/ml) of NaCl. The final bacterial concentrations were around 1 x 106 to 5 x 107 cells per ml. Samples of influent cell suspension and effluent samples were plated to quantify the number density of aerobic bacteria. In the case of microspheres and anaerobic C. acetobutylicum, direct microscopic counting with a hemacytometer was used. The pressure drop across the core holder was recorded by using a differential pressure transducer. The pumping rate was maintained at 40 ml/h throughout the experiment. The dimensions of the core were 3 in. long by 1 Suspensiona TABLE 1. in. in diameter (7.62 by 2.54 cm). Each experiment was repeated several times. Instrumentation. A constant flow high-performance liquid chromatography pump (model Constametric III manufactured by Laboratory Data Control, Riviera Beach, Fla.) was used in this experiment. The differential pressure gauge model P7D and signal demodulator model CD10 were purchased from Celesco Co., Canoga Park, Calif. Migration of bacteria through Berea sandstone containing nutrient medium. The apparatus (Fig. 4) consists of two flasks separated by a column of Berea sandstone rock wrapped in thermal shrinkage Teflon. After autoclaving at 125 C for 25 min, nutrient broth was aseptically poured into both flasks followed by a brief application of vacuum to one flask to remove entrapped air in the rock. Loop inoculation of one flask was done after the fluid levels across the rock had equilibrated. The development of turbidity in the other uninoculated flask indicated the migration of bacteria from the inoculated flask to the uninoculated flask. After the inoculated flask turned visibly turbid, we recorded the elapsed time, At, just when the other uninoculated flask became visibly turbid. The approximate migration rate was taken as the ratio of core length (-1.5 in. [3.81 cm]) to At. Sampling of both flasks was done for plating to obtain cellular density. B. subtilis and P. putida were used in this study. RESULTS The experimental conditions and the results are summarized in Table 1. The effluent history of injecting P. putida at a higher influent concentration of 5 x 107 cells per ml is shown in Fig. 5. The effluent concentrations decreased with time, and a rapid increase in pressure drop was observed. When the core was opened up at the end of the experiment, a layer of filter cake was seen outside the inlet surface of the core. However, when the influent concentration was lower (106 cells per ml), a different effluent history was observed (Fig. 6). No significant increase in the pressure drop was noticed. Also, no filter cake was found. Summary of core flooding experiments Calculated value (based on deep filtration model) Concn Porous mediumb Filtration Maximum retention capacity Species (cells/ml [inflow]) coefficient (cm-') (cells/ml; rock) P. putida (ATCC 12633) 5 x 107 CS C Microspheres 6 x 106 Oil-containing BS x 107 C. acetobutylicum spores 5 x 106 CS x 106 P. putida (ATCC 12633) 1 x 106 CS 0.61 d B. subtilis cells 1 x 106 CS 0.40 d B. subtilis spores 1 x 106 CS d a The flow rate is 40 ml/h for all experiments. A total of 1,000 ppm of NaCl is added to the carrier water. b BS, Berea sandstone; CS, Cleveland sandstone. The former has a permeability of 0.4 to 0.5 darcy, the latter around 4 darcy. c, Cannot be determined because plugging occurred and filter cake was formed. d, Cannot be determined because a short period of experimental time was involved. The inlet surface did not reach saturation at the end of the experiment.

5 1070 JANG ET AL. APPL. ENVIRON. MICROBIOL. - 5 x 10 7/ml 5 x 10/ml 3 X 105/mI I_ I-3.-.~ 4 X 104/ml I start 12 min. injection FIG. 5. The effluent history of injecting P. putida at a higher influent concentration into the Cleveland sandstone core, showing the effect of plugging and the formation of filter cake at the inlet surface. CL is the effluent bacterial count. It took about 12 min to pump 1 pore volume of suspension under the flow rate of 40 ml/h. The deep bed filtration coefficient of injecting spores of B. subtilis was much lower than with the injection of vegetative cells. Among the species tested, C. acetobutylicum spores have the lowest filtration coefficient and a measurable low retention capacity of 3 x 106 spores in 1 ml of bulk volume of the Cleveland sandstone core. The effluent history of injecting microspheres into an oil-containing Berea sandstone core is shown in Fig. 7. Contrary to the strong retention by the clean core as reported in reference 4, the breakthrough of microspheres was observed. CL -ci 106/mI CL - 104/mI FIG. 6. The effluent history of injecting P. putida at lower influent concentration. The inlet end was not yet saturated with cells at the end of experiment (about 2.5 h from the beginning of injection). CL is the effluent bacterial count. stort 12 min. 20 min h injection 1 FIG. 7. The effluent history of injecting negatively charged polymeric microspheres into an oil-containing Berea sandstone core. The presence of oil facilitates the transport of microspheres. CL is the effluent concentration of microspheres. In the experiments with microbial migration through a stagnant nutrient medium in the Berea sandstone core, the inoculated flask turned turbid within 6 h at room temperature. The uninoculated flask in the experiment with B. subtilis turned turbid at ca. 24 h (At) after the inoculated flask had turned turbid. Thus, the migration rate of B. subtilis can be taken to be ca. 1.5 in. (3.81 cm) per day. Plate count of samples from both flasks showed that as the cellular density reached 108/ml, the culture became visibly turbid. For P. putida, the elapsed time (At) and the migration rate were 48 h and 0.7 in. (1.78 cm) per day, respectively. DISCUSSION The experiments with P. putida indicated a potential danger of plugging the inlet surface of the core if the cells are injected at a higher concentration of 5 x 107/ml. P. putida at this concentration tend to form aggregates of 10 to 20 cells as observed by microscopy. The pores of the inlet surface were, therefore, easily plugged by the aggregates. Initial plugging in the inlet surface lead to the continued accumulation of cells from the influent. A decrease in the effluent concentration due to plugging is likely to occur. The injection of cells under this circumstance did not strictly follow the deep bed filtration model. The "deep filtration coefficient" calculated from the ratio of the influent concentration to the effluent concentration in this case is at best semiquantitative or qualitative (the first row of Table 1). The rapid increase in apparent deep filtration coefficient from 0.67 to 0.93 implied that pore plugging at the inlet surface occurred. The plugging of the influent surface not only increases the difficulty in injecting a bacterial

6 VOL. 46, 1983 suspension, but also hinders the transport of cells into the reservoir in the first stage of operation. From the practical point of view, the bench-scale study suggests that a high cell density for injection is not desirable. Also, bacterial species which show self-aggregating tendencies are not recommended for use in MEOR processes. The experiments with B. subtilis and P. putida at a lower injection concentration of 106/ml (row 4 to 6 of Table 1) showed a different pattern of effluent history (Fig. 6). The effluent history corresponded to the portion t < tmax of Fig. 2. The injection of cells in these cases followed the deep filtration model. The filtration coefficients are lower than that observed in injection at a higher concentration, indicating that cells are more easily pushed through the core at the lower injection concentration. Since only less than 20 pore volumes of bacterial suspension of P. putida and B. subtilis were injected in the experiments with lower influent concentration, probably the influent surface did not reach saturation at the end of each experiment. In other words, the maximum retention capacity Umax could have high values in these cases. In practice it is not necessary or economical to load a large amount of bacteria by injecting continuously a large number of pore volumes of dilute suspension or by injecting a concentrated cell suspension. As long as the nutrient supplied is sufficient, the further transport of bacteria after the initial distribution of cells provided by pumping can be achieved through in situ growth and migration. In the experiments with C. acetobutylicum spores at low injection concentration, not only is the deep filtration coefficient the lowest among all cases, but also the breakthrough of spores was observed. This implies that C. acetobutylicum spores are most easily pushed through the porous media among the species tested. Another advantage of injecting spores is their strength and rigidity, allowing tolerance to high shear rates and pressures encountered when the suspension is injected into the well. In addition, Clostridium spp. are gas, solvent, and acid producers which can find applications in MEOR processes. All the experiments with injecting bacteria were conducted with clean sandstone cores. The injection of negatively charged microspheres into the core containing residual oil did indicate that the presence of oil facilitated the injection of microspheres (Fig. 7), and a low filtration coefficient was observed with the breakthrough of microsphere. Can the presence of residual oil serve to facilitate the injection of bacterial cells? Our initial finding to this is positive (4). MICROBIAL-ENHANCED OIL RECOVERY 1071 By using the double-flask apparatus described, the migration of bacterial species through a section of porous rock can be easily observed. It would be possible to test many different bacterial species in a short time under various temperatures, pressures, and other medium conditions. Such experiments can assess the performance of potential bacterial species in the reservoir in the stage of static incubation after inoculation. Presently, work is being done to qualify bacterial passage through rock containing different inhibitors. The experiments with injecting a bacterial suspension and with the migration of viable cells through stagnant nutrient medium in the porous media were investigated independently in this work. To simulate the real transport in the MEOR processes, experiments with coinjecting cells (or spores) with nutrient followed by a period of static incubation are being scheduled. The results should reveal the relative transportability of various bacteria as the combined effects of pumping (stage 1) and migration (stage 2). The analysis in this paper provides a quantitative screening criterion for selecting proper potential bacterial strains for in situ MEOR applications based on the study of bacterial transport through porous rock. One might realize from the above analysis that no matter how efficient the metabolites secreted by certain bacterial species, the enhanced oil production rate is also affected by the transport of bacteria in the porous media. Therefore, it is worth devising methods to facilitate the transport of potential species which are strongly retained by the porous rock of the candidate reservoirs. ACKNOWLEDGMENT This work is supported by the U.S. Department of Energy under contract DE-AS19-81BC LITERATURE CITED 1. Bubela, B Role of geomicrobiology in enhanced recovery of oil: status quo. APEA J. B18: Herzig, J. P., D. M. Leclerc, and P. Le Goff Flow of suspensions through porous media-application to deep filtration. The 6th State-of-Art Symp. Div. Ind. Eng. Chem. Soc., June 9-11, 1969, Washington, D.C. Ind. Eng. Chem. 62(5): Jang, L.-K., J. E. Findley, and T. F. Yen Preliminary investigation on the transport problems of microorganisms in porous media. 28th IUPAC Meeting, Vancouver, B.C., Canada. Aug , p In J. E. Zajic, D. G. Cooper, T. R. Jack, and N. Korsaric (ed.), Microbial enhanced oil recovery, 1983, chapter 7. Penn Well Books, Tulsa, Okla. 4. Jang, L.-K, M. M. Sharma, J. E. Flndley, P. W. Chang, and T. F. Yen An investigation of the transport of bacteria through porous media, p International Conference on Microbial Enhancement of Oil Recovery, Shangri-La, Afton, Okla., May 16-21, In E. C. Donaldson and J. B. Clark (ed.), Proceedings, U.S. Department of Energy. Bartlesville Energy Technology Center, Bartlesville, Okla. (Available through National Techni-

7 1072 JANG ET AL. cal Information Service [CONF ) 5. Karaskiewics, J Recovery of crude oil from reservoir by use of bacteria. Nafta 24: Yarbrough, H. F., and V. F. Coty Microbially enhanced oil recovery from the upper cretaceous nacatoch formation, Union County, Arkansas, p International Conference on Microbial Enhancement of Oil Recovery, Shangri-La, Afton, Okla. May 16-21, In E. C. APPL. ENVIRON. MICROBIOL. Donaldson and J. B. Clark (ed.), Proceedings, U.S. Department of Energy. Bartlesville Energy Technology Center, Bartlesville, Okla. (Available through National Technical Information Service [CONF ].) 7. ZoBell, C. E Bacterial release of oil bearing materials. Part I. World Oil 126: ZoBell, C. E Bacterial release of oil bearing materials. Part II. World Oil 127:35-41.