Biotechnology in petroleum recovery: The microbial EOR Ramkrishna Sen , a,

aDepartment of Biotechnology, Indian Institute of Technology (IIT), Kharagpur, West Bengal 721302, India Received 17 January 2006; accepted 5 May 2008. Available online 20 June 2008.

Abstract Biotechnology has played a significant role in enhancing crude oil recovery from the depleted oil reservoirs to solve stagnant petroleum production, after a three-stage recovery process employing mechanical, physical and chemical methods. Biotechnologically enhanced oil recovery processes, known as microbial enhanced oil recovery (MEOR), involve stimulating indigenous reservoir microbes or injecting specially selected consortia of natural bacteria into the reservoir to produce specific metabolic events that lead to improved oil recovery. This also involves flooding with oil recovery agents produced ex situ by industrial or pilot scale fermentation. This paper essentially reviews the operating mechanisms and the progress made in enhanced oil recovery through the use of microbes and their metabolic products. Improvement in oil recovery by injecting solvents and gases or by energizing the reservoir microflora to produce them in situ for carbonate rock dissolution and reservoir re-pressurization has been enunciated. The role of biosurfactants in oil mobilization through emulsification and that of biopolymers for selective plugging of oil-depleted zones and for biofilm formation have been delineated. The spoil sport played by sulfate-reducing bacteria (SRB) in MEOR has also been briefly reviewed. The importance of mathematical models used in predicting the applicability of an MEOR strategy and the microbial growth and transport has been qualitatively discussed. The results of some laboratory studies and worldwide field trials applying ex situ and in situ MEOR technologies were compiled and interpreted. However, the potential of the MEOR technologies has not been fully realized due to poor yield of the useful microbial metabolic products, growth inhibition by accumulated toxic metabolites and longer time of incubation. A complete evaluation and assessment of MEOR from an engineering standpoint based on economics, applicability and performance is required to further improve the process efficiency for writing more success stories. Thus, this review attempts to address almost all the issues concerning the MEOR, its past and recent trends and its future prospect and directions. Keywords: Microbial enhanced oil recovery; Mechanisms; Solvents; Gases; Biosurfactants; Biopolymers; Biofilms; Selective plugging; SRB; Modeling; Field trials Article Outline

1. Introduction 2. Enhanced oil recovery 3. Microbial enhanced oil recovery 3.1. MEOR mechanisms 3.1.1. Role of biosurfactants 3.1.2. Role of biopolymers and biofilms 3.1.3. Use of gases and solvents as MEOR agents 4. Sulfate-reducing bacteriaNotorious villains in MEOR 5. Importance of mathematical modeling in MEORa qualitative analysis 6. Field trials 7. Conclusions Acknowledgements References 1. Introduction Crude petroleum is present worldwide in the complex capillary network of oil reservoirs. Traditional oil recovery technologies under the umbrella of chemically enhanced oil recovery (CEOR) can recover a maximum of 4045% of the oil initially in place, in two stages, namely, primary and secondary recovery. Thus, these conventional oil recovery operations often leave two-thirds of the oil in the reservoir. Primary recovery produces oil and gas using the natural pressure drive of the reservoir [1], [2], [3] and [4]. Secondary recovery involves stimulating the oil wells by the injection of fluids, which fracture the hydrocarbon-bearing formation to improve the flow of oil and gas to the wellhead. Several techniques

were used for injecting fluids into an oil reservoir to augment the natural forces in secondary recovery [4]. When water is the injection fluid, the process is called water-flooding. Injection of natural gas, which is an expensive option, constitutes an operation, called pressure maintenance [4] and [5]. Other techniques involving mechanical and physical means such as pumping and gas lift help in oil production when the reservoir pressure dissipates. While primary recovery produces 510% of the total reserve, recovery efficiencies in the secondary stage vary from 10% to 40% of the oil in place [3], [4] and [5]. To meet the rising energy demand worldwide, there is a dire need to produce more crude oil. Stagnant oil production and unimpressive recovery by conventional methods have been a major concern. The socio-political factors, which have become increasingly important in the post-Iraq war scenario, are adding fuel to this fire of concern. During the turbulent times of the Arab oil embargo, most major oil companies in the USA had set up their own research centers, and funded major programs to develop new technologies. These programs resulted in the creation of the enhanced oil recovery (EOR) technologies, which kept the industry going strong and competitive http://www.netl.doe.gov/scngo/Petroleum/Exploration%20&%20Production/EOR/eor.html . Hence, attention has been focused on the EOR techniques for recovering more oil from the existing and abandoned oilfields. Of these methods, microbial enhanced oil recovery (MEOR) has the potential to be cost-efficient [1]. MEOR refers to petroleum recovery methods, which involve the use of a mixed microbial population and the metabolic products including biosurfactants, biopolymers, biomass, acids, solvents, gases and also enzymes to increase recovery of oil from depleted and marginal reservoirs, thereby extending the life of the oil wells[1], [2], [3], [4] and [5]. Critical evaluation of the physical and biochemical mechanisms that control microbial responses to the hydrocarbon substrates and their mobility is a prerequisite for a comprehensive review on MEOR. Such reviews incorporating the in-depth analysis of the aspects of accession and transformation of hydrocarbons, changes in cell membrane architecture, cell-surface adhesion and hydrophobicity, chemotaxis, and also of the production and quality improvement of petroleum feedstock and petrochemicals have been presented [1], [3], [4], [5], [6], [7], [14], [15] and [16]. Thus, the purpose of this review is to discuss the operating mechanisms of MEOR and to highlight the recent developments and future prospects in this important area of scientific research. 2. Enhanced oil recovery The flow diagram (Fig. 1) shows the types of EOR processes that are currently employed in the oil industry. In the US alone the original oil in place has been estimated to be 650 billion barrels, a major part of which is the target of improved and advanced oil recovery methods (Fig. 2). Thus, more advanced technologies are being implemented in the oil industry today to recover the trapped oil under the program of EOR. In the US, the EOR methods including MEOR have the potential to recover much of that remaining oil, which is estimated to be about 375 billion barrels.

Full-size image (25K) Fig. 1. Flow sheet diagram showing the process steps in enhanced oil recovery (EOR).

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Fig. 2. Original oil in place in the USA (649 billion barrels), showing targets for EOR http://www.netl.doe.gov/scngo/Pe

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However, oil recovery is challenging because the remaining oil is often located in regions of the reservoir that are difficult to access and the oil is held in the pores by capillary pressure [6], [7] and [8]. No single EOR or MEOR approach can address this problem. A combinatorial strategy that involves the injection of a fluid or fluids to supplement the natural pressure in a reservoir, where the injected fluids interact with the reservoir rock/oil/brine system to create favorable conditions for maximum oil recovery, could be an efficient approach for the EOR [7] and [8]. The effect of capillary forces on trapping of oil within the pores of reservoir rock is normally characterized by the use of a dimensionless number, the capillary number. The capillary number is defined as the ratio of viscous to capillary forces as given in Eq. (1) [9] and [10]:

(1) where v and are the velocity and viscosity, respectively, of the displacing fluid, is the oilwater interfacial tension (IFT) and is the contact angle. The greater the capillary number, the lower the residual oil saturation in the reservoir and hence higher the oil recovery [10]. The normal microscopic view and a cross sectional view of a typical oil reservoir and the method of water-flooding are shown in Fig. 3.

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Fig. 3. A cross sectional view of an oil reservoir during water-flooding (adapted from http://www.titanoilrecovery.com/

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3. Microbial enhanced oil recovery Microorganisms, harmful and helpful, are ubiquitous in reservoirs around the world. These microbes are increasingly being acknowledged for their ability to influence reservoir behavior and oil mobilization [11]. The following are the oil recovery agents used for CEOR. Surfactants are used to reduce interfacial tension between oil and water, oil and rock interfaces. Polymers are used to increase viscosity of waterflood. Acids, gases and solvents are used to increase the permeability through the porous network and to re-pressurize the oil reservoir [1], [2], [3], [4], [5], [6], [7], [8], [10], [12], [13], [14], [15] and [16]. Since these are petrochemicals, obtained from petroleum feedstock after refining and downstream processing, CEOR methods turned out to be economically unattractive as the finished products are utilized for the recovery of raw materials. This is why the scientist looked for a cost-effective alternative and discovered the same in MEOR, which exploits microorganisms for the production of all the chemicals as mentioned above, albeit at a slower rate and with lower yields. In fact what is known today as MEOR finds its history dating back to 1947 [12]. Microbial consortia, as shown in Table 1, have been used in MEOR. They have the ability to produce biosurfactants, biopolymers, acids, gases and solvents to perform the job of recovering residual oil by fermenting cheaper raw materials, e.g. molasses [1], [2], [3], [4], [5], [11], [12], [13], [14], [15], [16], [17], [18], [21], [23], [24], [25], [26] and [27]. Biomass, which mostly secretes exopolysaccharides and forms biofilms, aids in MEOR by selective plugging of oil-less zones [1], [2], [3], [4], [27], [28], [29] and [30], as shown in Fig. 4. This has also been studied by nutrient resuscitation and growth of starved cells in sandstone cores [20]. Though MEOR is considered as an environmentally compatible tertiary oil recovery method and is a time tested and increasingly applied method of oil treatment in the industry as well, there is also growing application of microorganisms for the treatment of petroleum-based products both in reservoirs and on earth's surface for bioremedial cleanup of hydrocarbons [1], [3], [4], [5], [13], [16] and [19].

Table 1. Microbial consortia and their metabolites with applications in MEOR

Microbi al Example microbes produc t Biomass Bacillus, Leuconostoc, Xanthomonas Surfactan Acinetobacter, Arthrobacter, Bacillus, ts Pseudomonas Polymers Bacillus, Brevibacterium, Leuconostoc, Xanthomonas

Application in MEOR

Selective plugging and wettability alteration Emulsification and de-emulsification through reduction of IFT Injectivity profile and viscosity modification, selective plugging Rock dissolution for better permeability, oil viscosity reduction

Solvents Clostridium, Zymomonas, Klebsiella Acids Gases

Clostridium, Enterobacter, Mixed acidogens Permeability increase, emulsification Clostridium, Enterobacter Methanobacterium Increased pressure, oil swelling, IFT and viscosity reduction

Full-size table

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Full-size image (48K) Fig. 4. In situ MEOR processes (a) the thief zones are the targets for plugging by biomass or biopolymers, injected with

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MEOR scores over other EOR processes on two accounts. Firstly, the microbial cell factories need little input of energy to produce the MEOR agents and secondly, the application of microbial processes does not directly depend on the global crude oil price. In an in situ process, stimulation of the indigenous

microflora by injecting suitable nutrients serves to enhance oil mobilization. It is the exponential nature of microbial growth, which leads to the production of useful biochemical agents for MEOR at higher rates from inexpensive and renewable resources. However, there are a few shortcomings of MEOR. One major obstacle that has slowed the implementation of MEOR has been the difficulty in isolating and/or engineering microbial strains, which can survive in the extreme environment of the oil reservoirs. MEOR, to be economically viable, demands the use of microbial strains, which remain viable at such reservoir conditions as temperatures up to 85 C, pressure over 17.23 MPa, extremes of pH and salinity. Genetic engineering tools and techniques are being used to develop microorganisms that cannot only survive and grow in extreme reservoir environment, but can also subsist on inexpensive nutrients and produce substantial amounts of metabolic products as EOR agents including enzymes. Genetically engineered MEOR (GEMEOR) and the enzyme enhanced oil recovery (EEOR) constitute the advanced MEOR methods, which are not discussed here. 3.1. MEOR mechanisms The MEOR processes involve the use of reservoir microorganisms or specially selected natural bacteria to produce specific metabolic events that lead to enhanced oil recovery. The mechanisms by which MEOR processes operate can be quite complex and may involve multiple biochemical process steps [1], [2], [3], [4], [5], [6], [7], [8], [11], [12], [13], [14], [15], [16] and [26]. In selective plugging approaches, microbial cell mass and/or biopolymers plug high-permeability zones and lead to a redirection of the water-flood [27], [28], [29] and [30]. In other processes, biosurfactants are produced in situ which leads to increased mobilization of residual oil and reduces oil viscosity [1], [3], [5], [7], [8], [11], [26], [41] and [45]. In a MEOR process, conditions for microbial metabolism are supported via injection of nutrients. In some processes, this involves injecting a fermentable carbohydrate including molasses into the reservoir [11]. Some reservoirs also require inorganic nutrients as substrates for cellular growth or for serving as alternative electron acceptors in place of oxygen. A patented method involves injecting water containing a source of vitamins, phosphates and an electron acceptor such as nitrate into the formation and allowing anaerobic bacteria, either already present in or introduced into the formation to multiply using the oil as the main carbon source [25]. The mechanics of MEOR at a molecular level must be thoroughly understood to assess both the efficiency and the economic viability of the process. The microbes in MEOR are mostly hydrocarbon-utilizing, non-pathogenic and are naturally occurring in petroleum reservoirs [3], [4], [5], [16] and [22]. It is important to note that various Bacillus strains can produce the MEOR agents when grown on glucose mineral salts medium. These strains alone can be used when oil viscosity reduction is not the primary aim of the operation. But a consortium of both types of bacteria would be more useful and preferred. There are two major ways in which microbes may contribute to EOR: (a) they grow in reservoir rock to produce gases, biosurfactants, biopolymers and other non-toxic biochemical to recover trapped oil; and (b) they can selectively plug high-permeability channels so that the sweep efficiency of the recovery process can be increased (Fig. 4). It is of utmost importance to the petroleum industry that these microbial products should cause a series of very desirable changes in the physicochemical properties of the crude, and a marked improvement or a near-complete restoration of the lithological properties of the reservoir rock [3], [4], [5], [6], [7], [8], [11], [12], [13], [14], [15], [16],

[17], [22], [23], [24], [25] and [26]. Success of in situ MEOR operations depends on developing microbial consortia that can survive and produce the desired metabolic products in reservoirs containing hydrocarbons and saline water [5], [7] and [14]. Research activities are continuously focusing on anaerobic extremophiles including halophiles, barophiles and thermophiles for better adaptation to reservoir conditions [13], [14], [15], [16], [32], [41], [92], [93] and [94]. 3.1.1. Role of biosurfactants Surfactant EOR represents one of the most promising advanced methods to recover a substantial proportion of the residual oil [5], [7], [8], [9], [10], [11], [14], [15], [16], [22] and [26]. In this technology, an aqueous surfactant formulation is injected into a mature oil reservoir. Where this solution contacts the small blobs of oil trapped in the pores of the reservoir rock, it dramatically reduces the interfacial tension (IFT) and mobilizes this trapped oil by increasing the capillary number [7], [8], [9], [10], [11], [26], [31], [32], [33] and [34]. Formation of an oil-in-water emulsion often leads to an improvement in the effective mobility ratio until the surfactant is diluted or otherwise lost due to adsorption on the rock. The core floods conducted in Berea sandstones using Yates stocktank oil and synthetic brine showed the ability of these surfactants to improve wettability for enhanced oil recovery [9] and [10]. Surfactants thus contribute positively to improve oil recovery by reducing IFT and also by altering the wettability of reservoir rock for water-flood to displace more oil from the capillary network. The Department of Energy http://www.netl.doe.gov sponsored projects at the California Institute of Technology to design new surfactant systems to make surfactant EOR process commercially attractive. Another project sponsored by DOE was to employ advanced biotechnology methods to enhance biosurfactant production from selected bacterial strains acclimatized to reservoir conditions. As biosurfactants play a major role in MEOR [5], [7], [8], [9], [10], [13], [14], [15], [16], [26], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40] and [41], production and application aspects of rhamnolipid, sophorolipid, glycolipid and lipopeptide biosurfactants assume great industrial significance and thus have been extensively reviewed in literature [42], [43], [44] and [45]. Biosurfactants with potential applications in MEOR are listed in Table 2. Though glycolipid biosurfactants produced by Pseudomonas strains have been extensively used in MEOR experiments, lipopeptides like surfactin, lichenysin and lipid polysaccharide complexes like emulsan have also been found very effective. Surfactin, a lipopeptide and the most potent microbial surfactant known so far, has been produced ex situ in the controlled environment of a fermenter and has been reported to be successfully used in MEOR [35]. A bioprocess was developed and optimized for the enhanced surfactin production in three stages, namely, optimization of the nutritional factors in the production medium [46], optimization of the environmental parameters including pH, temperature, rates of agitation and aeration [47] and optimization of the inoculum age and size for judicious dosing of seed culture [48]. The concentration and purification parameters of surfactin when recovered by ultrafiltration method in one step after broth clarification have been characterized and reported recently [49].

Table 2. Various potential microbial surfactants for application in MEOR Biosurfactant and microbial source Reference Source: Bacillus sp. Surfactin Rhamnolipid Lichenysin Schaller et al. [35] Banat [26] Jenneman et al. [31] McInerney et al. [33] Yakimov et al. [34] Horowitz and Griffin [36] Source: Acinetobacter sp. Emulsan Alasan Source: Pseudomonas Rhamnolipid Source: Rhodococcus sp. Viscosin Trehaloselipids Full-size table Neu et al. [40] Tango and Islam [41] Arino et al. [39] Rubinovitz et al. [37] Navon-venezia et al. [38]

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Usually, the biosurfactants, which are produced in fermenters ex situ, are injected with other chemicals in the water-flood to facilitate the emulsification of oil with the water jet for better recovery. However, in situ production and application of biosurfactants in a limestone reservoir has recently been reported [95]. Experimental studies have been performed to assess the potential of biosurfactants for their applications as MEOR agents [22], [26], [31], [32], [34], [45], [50], [51], [52], [53] and [54]. In order to

show the efficacy of some of the biosurfactant producing strains, the results of laboratory scale simulated experiments on emulsification index and percentage of oil recovered from sand packed columns are presented in Table 3. The emulsification index (E24) was determined by adding 6 ml crude petroleum to 4 ml culture broth in a graduated tube, followed by vortexing at high speed for 2 min. The emulsion stability was determined after 24 h. The emulsification index is thus defined as the height of the emulsion layer, divided by the total height, multiplied by 100.

Table 3. Results of laboratory scale simulation experiments are given Emulsification index (E24) 8090 % Oil recovered from sand packed column 90100 62 56 82 90 8794

Microbial strain

References

Bacillus sp. AB-2

Banat [50] Makkar and Cameotra [51] Makkar and Cameotra [52] Pruthi and Cameotra [53] Pruthi and Cameotra [54] Sen (unpublished data) [55]

Bacillus subtilis MTCC 2423 90 Bacillus subtilis MTCC 1427 33.3 Serratia marcescens 94

Arthrobacter protophormiae 60 Bacillus subtilis DSM 3256 Full-size table 5763

The glass column that was used for surfactin from B. subtilis DSM 3256 was 3612.5 cm in dimension and was packed w before being used for the oil recovery experiment. Crude surfactin (approximately 1 g/L in broth) was used in this stud

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3.1.2. Role of biopolymers and biofilms Biopolymers have been used in MEOR experiments mainly for selective plugging of oil-depleted zones and hence, for permeability modification [3], [5], [7], [11], [15], [18], [20], [21], [22], [23], [24], [30], [57], [58], [59], [60], [61], [62], [63], [64], [65] and [66]. In selective plugging approaches, biopolymers plug high-permeability thief zones to redirect the water-flood to oil-rich channels. This is an adjunct to waterflooding operations, in which water is pumped into injection wells in the reservoir in order to force the oil up to the surface bypassing the oil-depleted zones in the reservoir [23], [27], [30], [32] Illias RM, Ooi

SW, Idris AK, Rahman WA. Production of biosurfactant and biopolymer from Malaysian oil fields isolated microorganisms. SPE J 1999; Paper no. 57315.[32], [57], [58], [59], [60], [61], [62], [63], [64], [65] and [66]. Bacterial plugging usually involves the feeding of injected or in situ bacteria within the reservoir [3], [5], [7], [11], [21], [23], [27] and [30]. Bacteria and/or nutrients preferentially enter the reservoir along high-permeability pathways. Biomass growth in those laminae plugs the pore throats, thus decreasing the permeability in what had once been the high-permeability zones. This tends to equalize the permeability across the reservoir and restore the sweep efficiency of water-flooding operations [5], [7], [11], [12], [13], [14], [15], [16] and [21]. Some biopolymers that have potential applications as mobility control agents in MEOR are listed in Table 4.

Table 4. Exopolysaccharide biopolymers used in EOR with their microbial sources [57], [58], [59], [60], [61], [62], [63], [ Biopolymer and microbial sp. Reference

Xanthan gum (Xanthomonous sp.) Pollock and Thorne [59] Pullulan (Aureobasidium sp.) Levan (Bacillus sp.) Curdlan (Alcaligeness sp.) Dextran (Leuconostoc sp.) Scleroglucan (Sclerotium sp.) Full-size table Cho et al. [62] Akit et al. [63] Buller and Vossoughi [64] Kim and Fogler [65] Sandford [66]

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Selective plugging was initially thought to have occurred primarily due to sieving action which is best achieved when the average pore throat radius is less than twice the diameter of the bacteria [71]. Dead bacteria or non-slime producing bacteria either do not plug porous media, or plug the media to a lesser extent than do similar strains that produce an exopolysaccharide layer. The polysaccharides secreted by many strains of bacteria serve mainly to protect the bacteria against desiccation and predation, as well as to assist in adhesion to surfaces [3], [7], [13], [14], [15], [21], [65] and [71]. The effects of biopolymers on fluid conductivity in sand columns and sandstone permeability have been well studied in laboratory trials [17], [18], [27], [30], [64], [65], [66], [67], [68] and [69]. As plugging and mobility control agents,

biopolymers are perhaps more efficient than the bacterial bodies. Application of these processes in field trials has made the feeding program effective in slowing the deterioration of hydrocarbon in wells undergoing water-flooding operations by diverting the water jet from high-permeability zones to oil-rich zones [1], [2], [3], [4], [5], [6], [7], [8], [11], [12], [13], [14], [15], [16], [23], [27], [30], [64], [65], [66], [67], [68], [69], [71], [72], [85], [86] and [87]. The use of bacterially produced polysaccharides as floodwater thickening agents is well known. Xanthomonas species has been used to ferment carbohydrates and produce a thermally stable heteropolysaccharide called xanthan gum. Xanthan gum has been used in water-flooding systems. Though xanthan gum is an ex situ product of fermentation of carbohydrates, the use of it comes under chemical enhanced oil recovery. The efficacy of this biopolymer as an oil recovery agent has been studied by injecting an aqueous mixture of the polysaccharide (xanthan gum) and sodium chloride in a model reservoir in the laboratory [57], [68] and [69]. Processes based on the uses of xanthan gum in EOR involve thickening the injection waters; using the biopolymer with other chemicals or adsorption agents to prevent adsorption of the polysaccharide on to rock surfaces; using multivalent cations to bind the polysaccharide molecules together for preferential partial plugging and applying with gel particles, like hydrolyzed polyacrylamides, and ferric chloride solutions to facilitate the thickening process [56], [58] and [85]. Its physical properties of viscosity, shear resistance, temperature and salt tolerance make it almost an ideal polymer for use in EOR [59], [60] and [61]. It is considered superior to polyacrylamide for the above-stated reasons. However, it is more expensive and more susceptible to bacterial degradation [67]. Another biopolymer, scleroglucan, is produced by a species of the fungus Sclerotium and was examined for possible use in EOR [66] and [70]. Biopolymer levan was found to be potentially suitable in oil reservoirs which have a temperature of less than 55 C, a pH between 6 and 9, a pressure less than 500 atm and a salt concentration of 4% [63]. Though xanthan gum has been the most effective biopolymer, applications of other biopolymers like curdlan, dextran and pullulan in MEOR have also been reported [1], [3], [4], [5], [11], [62], [64], [65] and [66]. On the other hand, biofilms are heterogeneous systems of bacteria, their exopolysaccharides and water channels. Studies on biofilms indicated that they were composed of less than 27% bacterial bodies and the remainder (7398%) was assumed to be composed of extracellular products, probably exopolysaccharides and void space [69], [71], [72] and [85]. Biopolymers may occur in several different morphologies forming biofilms within porous media like oil reservoirs. Within the porous media, the morphology may assume a web like structure, called bioweb at a certain stage of growth [72]. Biopolymer production and consequent biofilm formation are both important for MEOR operations to be effective. These two phenomena are influenced by such parameters as water chemistry, pH, surface charge, microbial physiology, nutrients and fluid flow [3], [66] and [71]. 3.1.3. Use of gases and solvents as MEOR agents The use of bacterial consortia to produce gases, solvents and acids for enhancing oil recovery by exploiting the mechanisms of reservoir re-pressurization and carbonate rock dissolution has been an old practice [1], [12], [14], [15], [68], [71], [106] and [114]. The microbial consortia and the operating mechanisms are enlisted in Table 5. Bacteria can ferment carbohydrates to produce gases such as CH4,

CO2 and H2. Such gases produced in situ can contribute to pressure build-up in a pressure-depleted reservoir. These gases may dissolve in the crude oil and reduce its viscosity. Additional benefits of this bacteria-induced fermentation process include the production of acids, such as acetic and propionic acids and the production of solvents, such as acetone, ethanol, 1-butanol and butanone. Both gases and solvents can dissolve the carbonate rock, thereby increasing its permeability and porosity; anaerobic bacteria, Clostridium acetobutylicum, Enterobacter cloacae and Methanobacterium have been the microbes of choice for their availability as indigenous natural reservoir microflora. Extensive research has been carried out in understanding the biofilm growth in porous media to harness indigenous microorganisms that promote increased oil recovery from depleted oil reservoirs at low cost.

Table 5. Solvents, acids and gases with their producing microorganisms and MEOR mechanisms Solvents, acids and gases with their microbial sources Acetone, butanol, propan-2-diol (Clostridium, Zymomonas, Klebsiella) Propionic and butyric acids (Clostridium, Enterobacter, mixed acidogens) Methane and hydrogen (Clostridium, Enterobacter, Methanobacterium) Full-size table

Operating mechanisms

References

Improved permeability by carbonate rock dissolution and oil viscosity reduction Enhancement of permeability and degree of emulsification

[12], [14], [68] and [71] [15], [57], [68] and [71]

Rock re-pressurization, oil swelling, reduction [57], [68], [71] of IFT and oil viscosity and [106]

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4. Sulfate-reducing bacteriaNotorious villains in MEOR The sulfate-reducing bacteria (SRB), which have relatively simple growth requirements in sulfate and carbon as energy sources, play a very negative role in MEOR [11] and [78]. SRB are traditionally known to be active in shallow wells, but the existence of significant SRB populations in deep, hot and highpressured reservoirs has been confirmed from souring of the formations. The association of the onset of reservoir souring, which jeopardizes the economical calculations, with the starting of seawater injection in previously so-called sweet fields is largely due to SRB activities [11] and [78]. The SRB are extremely sturdy and can survive prolonged starvation in seawater at both surface and reservoir temperatures. In this dormant state, starved SRB tend to be smaller in size than their growing counterparts and may

travel longer distances through the porous network during water-flooding. Chemicals, such as biocides, are regularly injected as slug doses in an intermittent treatment program to kill SRB. But starved SRB are less affected by standard biocide treatments than actively growing populations [3], [5], [11] and [78]. The continuous biogenic sulfide contamination of huge rock formations and production facilities has belied the theory that microorganisms cannot significantly influence the micro-environment of the oil reservoirs [73]. The major concerns of the global oil industry include souring, corrosion caused by H2S, plugging by iron sulfide, the related financial burden and the threat to health and safety of the operators. The so-called Bio-Competitive Exclusion (BCX) technology http://www.ptac.org/res/dl/rest0302p.pdf exploits this potential and targets indigenous de-nitrifying bacteria (DNB) and manipulates the entire reservoir microflora to release trapped oil for commercial production, as well as inhibit the sulfide production by SRB almost permanently [89]. This oil-mobilizing and sulfide-removing microbial system was first introduced to industry as Max-Well 2000, which has proven its efficacy in the real time field tests involving the injection of low-cost nutrients [73]. There are reports on the effects of hydrocarbon utilizing SRB on corrosion of metallic parts of the drilling pipes in injection and production wells and also on nitrate-mediated microbial control of reservoir souring on the efficiency of MEOR [74] and [75]. The dynamics of microbial control of H2S production in oil reservoirs has been elucidated [76]. The control of iron and H2S has been tested in an oil field by employing BCX process [77]. 5. Importance of mathematical modeling in MEORa qualitative analysis Structured mathematical models are required to describe the MEOR processes in a better way. In order to develop a proper field strategy, formulation of an efficient reservoir simulator capable of predicting bacterial growth and transport through porous network and the in situ production and action of the metabolites called MEOR agents is of paramount importance. Several studies have been reported on mathematical modeling of biomass growth, selective plugging and bacterial transport in porous media [79], [80], [81], [82] and [83]. The simplified models were developed based on fundamental conservation laws along with growth, retention kinetics of biomass and biomass concentration in aqueous and oil phases in order to predict porosity reduction as a function of distance and time or based on a filtration model to express bacterial transport as a function of pore entrance size and also to relate permeability with the rate of bacteria penetration by applying Darcy's law [80], [81] and [82]. From an order of magnitude analysis, it is known that only biosurfactant flooding and selective plugging mechanisms have the potential to recover more oil in MEOR process. This is the major reason behind the formulation of mathematical models of MEOR process based on these two mechanisms. The basic equations governing the transport of oil, water, bacteria, nutrient and metabolites in MEOR are based on component mass balance and overall mass conservation. The biomass-plug propagation and channel breakthrough using Bingham yield stress of biofilm were described well by a biofilm removal model, which represents the stability of biofilm against shear stress. The simulation results showed that the biofilm models based on Bingham yield stress predicted the biomass accumulation and channel breakthrough reasonably well [81]. A three-phase, multiple-species, one-dimension model was developed to simulate microbial growth and transport, and metabolism

processes involved in MEOR and to predict permeability modification that results from these microbial activities in porous media. Convection dispersion equations and microbial kinetics were incorporated in the model system to characterize and quantify biomass production, product formation, and nutrient utilization in the MEOR process [82]. The model was validated by applying to static (sand packs) and core-flooding (sandstone cores) experiments to describe microbial movement, metabolite production, and nutrient consumption during growth and metabolism and also to estimate permeability reduction. This could be extended to provide numerical predictions for the purposes of design and evaluation of MEOR field projects [82]. Some of the relevant scaling up criteria and numerical simulation results were discussed in order to explain the difficulties of scaling up laboratory results before planning a field application [83]. The experimental conditions of the MEOR technique applied for Garzan oil (26 API; southeast Turkey) were utilized in a mathematical model that describes the transport of bacteria and its nutrients by convective and dispersive forces, including bacterial decay and growth [84]. A recent study that generated essential data for modeling in situ MEOR processes included specific growth rates, carbon balances, biosurfactant production rates and biosurfactant yields [95]. But a more comprehensive approach based on artificial neural network modeling would be more useful to develop suitable models for describing the in situ MEOR processes. 6. Field trials Microbial enhanced oil recovery technologies have progressed from laboratory-based studies in the early 1980s to field applications in the 1990s. The oil reservoirs worldwide represent very complicated biological systems for which laboratory simulations of microbial activities become very challenging. The microbial consortia that are introduced into an oil field would have to compete with the indigenous microflora. While it may be possible to show beneficial effects in laboratory conditions with tailored microbial cultures, in the reservoir these are likely to be out-competed by the better adapted indigenous species. In most of the cases, success or failure of field applications of microbial technology is often hindered by the absence of specific and quantitative understanding of microbial activity. A reservoir engineering perspective, focusing on issues such as scale-up of laboratory results, process design, and field implementation and operation provides a consistent framework for comparing MEOR with other EOR processes [87]. This justifies the rationale behind the pilot scale field trials that are undertaken after successful development of MEOR processes through R&D in laboratory settings. Field trials to determine and document the effectiveness of microbial processes and to assess the validity of laboratory studies and models have been conducted. The application of MEOR in these trials has resulted in a substantial and sustained increase in production compared to control operations on the same reservoir [88]. A pictorial presentation of the state of the oil droplets in a porous sandstone rock before and after the MEOR process is illustrated in Fig. 5.

Full-size image (66K)

Fig. 5. Before MEOR process: oil is trapped in porous sandstone. Water flows without dislodging oil droplets from tiny p particles in the reservoir for enhanced recovery (adapted with permission from http://www.titanoilrecovery.com/titan

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In the last decade of the last millennium, a number of MEOR field projects have been conducted in various parts of the world with varying degrees of success. The field assessment of a MEOR technology applied in the Alton field in Australia showed an approximate 40% increase in net oil production, 12 months after treatment, which sustained for considerable period of time [88]. Microbial enhanced water-flooding technology has also been shown to be an economically feasible technology in the United States [16]. Deliberations on information and papers presented by companies and researchers all over the world on field applications using microbes and also the details of MEOR projects have been in public domain [11] and [16]. A compilation of very relevant information and data of field trials conducted in the USA and Romania is presented in Table 6.

Table 6. Details of some field trials showing the efficacy of MEOR methods (adapted from Bass and Lappin-Scott [11]) Field test details Year of initiation Oil field Formation Depth (m) Permeability Phoenix Pilot Project, NIPER SE Vasser Vez Sand Pilot Project, OU Institute of Biology, Romanian Academy

1990 Chelsea-Alluwe Bartlesville sandstone 122 16

1991 SE Vasser Vertz sand unit Vertz sandstone 550 60181

1990 Bragadiru Information not available 780 150300

Field test details (md) Salinity (%) Oil viscosity (cp)

Phoenix Pilot Project, NIPER

SE Vasser Vez Sand Pilot Project, OU

Institute of Biology, Romanian Academy

2.9 6

1119 2.9 5 treated 19 Molasses, NH4NO3

0.060.3 9 1 treated Information not available Molasses Bacillus, Clostridium, Arthrobacter, Pseudomonas, Micrococcus Biosurfactants, biopolymers, acids, gases and solvents Yes 4 Years 1.5 B/D per well

Injection wells 19 Production wells 47

Nutrients used Cane molasses

Microorganism Bacillus, Clostridium Indigenous microflora (s) MEOR-Agents Water flood Test length Pre-MEOR production Post-MEOR production Comments Biosurfactans, acids Biomass and gases and gases Yes 1.5 Years 1 B/D per well Yes Approximately 9 months No oil production before injection 83 barrels produced (January 92June 92)

1.2 B/D per well

7.4 B/D per well Cyclic microbial recovery, well-bore clean up

20% increment in oil Decreased permeability production

Full-size table NIPER: National Institute of Petroleum and Energy Research, USA; OU: Oklahoma University.

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An analysis of the data from a data base of information collected from 322 projects employing the same MEOR methods resulted in the evaluation of the technical effectiveness and process economics of this particular technology and provides a source of information useful for predicting treatment response in any given reservoir [89]. The application of MEOR in these trials has resulted in a substantial and sustained increase in production compared to control operations on the same reservoir. Obviously, there are differences in the extent of improvement in oil recovery, which is influenced by various factors including individual reservoir characteristics like lithology, nature of the sands, porosity, permeability, reservoir temperature, crude oil gravity and the drive types. In addition to the bottom-hole conditions and the nature of the formations, bacterial composition and concentration of the inocula, their adaptation time, optimum slug size and the application time constitute the major governing factors in MEOR. Efforts to explain these differences are severely limited by the lack of quantitative measures of microbial performance in terms of the reaction rates, stoichiometry, required product concentrations, etc. It is possible, however, to demonstrate quantitative relationships between microbial performance, reservoir characteristics and operating conditions [87]. An unfortunate consequence is that the relevance of microbial performance to reservoir engineering design is often obscure. Conversely, explaining success or failure of field applications of microbial technology is often hindered by the absence of specific and quantitative understanding of microbial activity. In an MEOR field study in the Southeast Vassar Vertz Sand Unit salt-containing reservoir in Oklahoma, nutrient injection stimulated growth of the microbial populations, thereby decreasing the effective permeability by 33% [90]. A biosurfactant flooding process using a very low concentration (3541 ppm) of biosurfactant produced by Bacillus mojavensis strain JF-2 has been recently reported to be very effective in recovering about 3545% residual oil from Berea sandstone cores [91]. Literature reports discussing in situ applications of MEOR in field trials with analysis of the results are available [17], [18], [89], [92], [93], [94], [95] and [98]. Very recently, a field study involving in situ production and application of biosurfactants by a consortium of Bacillus strains demonstrated that approximately nine times the minimum concentration of biosurfactant required to mobilize oil was produced in situ and resulted in the recovery of substantial amount of oil entrapped in the limestone reservoir of the Bebee field, Pontotoc City, Oklahoma, USA [95]. The technical and economical feasibility studies of MEOR were carried out in mature water-floods in continuation of a pilot study of controlled microbial colonization in producer wells in La Ventana oil field of Argentina [96]. The MEOR pilot projects carried out in Providencia and Lobitos oil fields in Peru resulted in 36.5% and 46.5% increments in oil production, respectively, and were considered profitable and economically feasible of further expansion [97]. A new MEOR technology, termed as microbial permeability profile modification (MPPM) technology, has been reported to improve oil recovery by adding nitrogenous and phosphorus-containing nutrients to the injection water of a conventional waterflooding operation [99]. This MPPM technology has been claimed to have extended the economic life of the field called North Blowhorn Creek Oil Unit (NBCU) located in Alabama by 60137 months, with an expected recovery of (46)105 bbl of additional oil. Another microbial profile modification method employing a spore-bearing halotolerant mesophilic bacterium has been reported. This bacterium produces spores that can propagate easily in Berea cores with permeability >500 md and can reduce the

rock permeability by producing biofilm on germination under suitable nutritional and environmental conditions [100]. Economically attractive alternative EOR technologies including microbial EOR, have been discussed and numerical simulation results have been presented to demonstrate the difficulties in scaling up and translating the laboratory results to real-time oil field settings [101]. Comprehensive R&D studies and annual reports on the development of microbial strains with improved transport and biosurfactant activity for EOR and on the biosurfactant-mediated oil recovery in model porous systems with its computer-aided simulations have tremendously contributed to the design and development of effective MEOR strategies and at the same time have enormously enriched the scientific literature on MEOR [102], [103] and [104]. Applications of MEOR processes in the oil fields in Asian countries, for example in Chinese, Malaysian and Indian oil fields, have been reported [92], [105], [106], [107], [108], [109] and [110], though published literature does not have field trial data in an organized format as shown in Table 6. In Malaysia, the estimated oil-in-place from the 47 producing fields stands at about 20.1 BSTB (billion stock tank barrels), with a cumulative production of 4.9 BSTB till 2003 [105]. However, there were no published reports on full-fledged field application of MEOR in Malaysia, with the exception of an MEOR stimulation project in Bokor field [105]. There is one structured report on MEOR field trial in the Fuyu oil field, Jilin province, China. The trial was carried out in 2001 in the sandstone formation with an average permeability of 180 md at a depth of 300500 m and an oil viscosity of 68 cp. The recovery of oil could be doubled after 6-month MEOR treatment from 14 production wells by injecting a bacterial consortium containing Enterobacter cloacae and Bacillus licheniformis as major bacterial strains and a nutrient package mainly comprising of molasses (10%) and cellulase enzyme (0.01%) [106]. Microbiological and chemical characteristics of the production fluids of the high-temperature Liaohe oil field in China were examined to investigate the potential for MEOR [107] and [108]. It was concluded that the Liaohe oil field contained a diverse thermophilic microbial community having a high biotechnological potential for petroleum recovery [107]. Recently, a study to assess the potential applicability of MEOR techniques in the Kongdian reservoirs of the Dagang oilfield in China showed that the oil field was inhabited mostly by anaerobic thermophilic fermentative, sulfate-reducing and methanogenic bacteria and hence, had great potential for application of MEOR processes by activating the stratal microflora [109]. In an exploratory study with the formation water from Daqing oil field, China indicated that the oil field was found to be inhabited by aerobic bacteria like Bacillus, Pseudomonas, Rhodococcus, Dietzia, Clavibacter, etc., which were capable of producing effective oil-releasing agents including biosurfactants [110]. A laboratory study involving growth of some selected strains in the crude oil samples and consequent reduction of oil viscosity indicated that the results could be translated into an effective strategy for recovering residual oil from Daqing oil field, the biggest oil field in China [111]. The MEOR squeeze treatment, which was carried out for about 3540 days in 1997, was found to have increased production performance and decreased water cut in 17 wells out of 25 wells treated. Another laboratory study using a model reservoir simulating the conditions of Daqing oil field and a pilot study in some wells of the same oil field employed the metabolic products (PIMP) from a strain of Pseudomonas aeruginosa, which showed significant increment in crude oil recovery and prolongation of the cycle of oil well washing to increase total oil production [112].

In India, the Oil and Natural Gas Corporation (ONGC) Limited, in collaboration with The Energy and Resources Institute (TERI, New Delhi) and the Institute of Reservoir Studies (IRS), Ahmedabad, conducted some field trials by employing a Huff and Puff process and using an indigenously developed MEOR technology based on a consortium of anaerobic extremophiles isolated from the candidate reservoirs. A three-fold increase in oil production and a significant reduction in water cut were achieved by applying this technology in 9 wells out of 12 wells treated in 4 oil fields, mostly in the state of Gujarat [113]. Recently, a US patent has been granted on the same process and microbial consortium, which was developed in TERI in collaboration with IRS and was evaluated in field trials by ONGC [114]. 7. Conclusions MEOR represents a truly eco-friendly petroleum recovery process employing biotechnological resources and techniques that can be used to replace and augment the traditional EOR processes and flooding chemicals. Many countries have envisaged that one-third of their oil recovery programs will utilize MEOR techniques by the year 2010. Though it has been repeatedly encountered that the benefits of MEOR projects applied to one well have positively affected the recovery performance of neighboring wells, thus improving the process efficiency; better understanding of the reservoir characteristics, biochemical and physiological characteristics of microbial consortia, controlling mechanisms and process economics are essential before MEOR becomes a viable process for general field applications. Most of the MEOR work leading to field trials has been completed in the last two decades and now the technology has advanced from a laboratory-based evaluation of microbial processes, to field applications internationally. Though research initiatives and endeavors in MEOR have progressed rapidly, still some questions remain unanswered. In spite of microbial processes holding great promise and prospect for EOR, the reasons that retard the implementation of MEOR include inconsistency in in situ performance, low ultimate oil recovery factor, uncertainty about meeting the engineering design criteria by microbial processes and a general apprehension about processes involving live bacteria. It is now known that manipulation of microbial consortia activity is a potentially powerful process that can profoundly and beneficially affect the microenvironment of a reservoir to mobilize more oil, increase production rates and technical efficacy of the recovery systems. In situ application of a judicious consortium of aerobic and anaerobic bacteria, acclimatized in simulated oil reservoir conditions, coupled with the bioaugmentation and stimulation of indigenous microflora have been successful in enhancing both the oil productivity and ultimate oil recovery factor. However, a better understanding of the MEOR processes and mechanisms from an engineering standpoint based on economics, applicability and performance is the key to further improve the process efficiency for writing more success stories. Advanced MEOR technologies based on the use of genetically engineered organisms and some specific enzymes may prove to be very effective and successful in the future. Nevertheless, an urge and urgency in developing novel MEOR technologies in the wake of an impending energy crisis seem to be imminent, mainly on account of their perceived potential to offer a cost-effective solution to the conventional EOR strategies. Acknowledgments

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