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Production-related petroleum microbiology: progress and prospects

Gerrit Voordouw
Microbial activity in oil reservoirs is common. Methanogenic consortia hydrolyze low molecular weight components to methane and CO2, transforming light oil to heavy oil to bitumen. The presence of sulfate in injection water causes sulfatereducing bacteria to produce sulde. This souring can be reversed by nitrate, stimulating nitrate-reducing bacteria. Removing biogenic sulde is important, because it contributes to pitting corrosion and resulting pipeline failures. Increased water production eventually makes oil production uneconomic. Microbial fermentation products can lower oil viscosity or interfacial tension and produced biomass can block undesired ow paths to produce more oil. These biotechnologies benet from increased understanding of reservoir microbial ecology through new sequence technologies and help to decrease the environmental impact of oil production.
Address Petroleum Microbiology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T21 1N4, Canada Corresponding author: Voordouw, Gerrit (voordouw@ucalgary.ca)

consider how to use this resource with the least degree of environmental harm. The quality of a primary energy source can be indicated by its energy returned on energy invested (EROEI), usually abbreviated as EROI [2]. The EROI for the best Middle-East reservoirs is estimated at 100:1. Values for deep offshore oil or for the Alberta oil sands are lower, due to increased energy input needed for production. Because these represent an increasing fraction of total production, the overall EROI value for world oil production is sliding. A goal of production-related petroleum microbiology should therefore be to decelerate the speed of the slide. Recent contributions, concrete or potential, to this effect are presented here.

Oil conversion by methanogenic consortia

Selected oil components are hydrolyzed to methane and CO2 by consortia of hydrocarbon-degrading syntrophic bacteria (syntrophs) and methanogens [3]. The syntrophs convert oil components to either acetate or H2 and CO2, which is thermodynamically uphill unless methanogens are present [3,4]. Water is present in sedimentary rock below the oil in the zone of oil-water contact, where preferential use of low molecular weight components converts light oil to heavy oil to bitumen [4,5]. Both hydrogenotrophic [5,6] and acetotrophic methanogens [7] have been found in these consortia [8]. Many syntrophs belong to the deltaproteobacteria (e.g. the genus Syntrophus), the same class to which many SRB belong. In the absence of sulfate these participate in syntrophic consortia and this may also apply to heterotrophic nitrate-reducing bacteria (hNRB). The scarcity of electron acceptors in oil elds imposes the need to participate in syntrophic methanogenic consortia, switching to more protable electron acceptor-dependent metabolism when possible. Thermophilic methanogenic enrichments from an Alaskan oil eld converted up to 50% of C9-C31 n-alkanes and lower fractions of aromatic hydrocarbons into methane and CO2 [9]. The process is not limited to oilelds. Oil sand tailing ponds can emit up to 107 L of methane per day from conversion of naphtha hydrocarbon diluent used in bitumen extraction [10]. Water-containing oil storage tanks can also evolve signicant methane [11]. Methanogenic hydrocarbon degradation offers the potential to convert oil that can no longer be economically produced to methane [12]. However, abundant availability of gas from subsurface shales and higher prices for oil as
Current Opinion in Biotechnology 2011, 22:15

Current Opinion in Biotechnology 2011, 22:15 This review comes from a themed issue on Environmental biotechnology Edited by Lindsay Eltis and Ariel Kushmaro

0958-1669/$ see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.12.005

Introduction
An age-long challenge has been how to gain and retain access to an adequate source of energy. The primary energy supply of the day tends to become depleted and its wide-scale use invariably causes environmental harm, forcing consideration of alternatives. Use of wood led to strong declines in forest cover in Europe from BC2000 to AD1800 [1], when this primary energy supply was nally supplanted by coal and then by oil and gas. The current environmental problems associated with the wide-scale use of fossil fuels are well-known, increasing the desire for more sustainable alternatives. However, the scale of our energy use makes it hard for any alternative to ll fossil fuels large size boots. Hence, fossil fuels will be with us for many years to come making it imperative to
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2 Environmental biotechnology

Table 1 Survey of petroleum microbiology based biotechnologies relevant to oil production. Biotechnology Conversion of oil to methane Nitrate injection to remove sulde from high temperature reservoirs Nitrate injection to remove sulde from low temperature reservoirs Status Experimental Mature Mature/in progress Factors hindering implementation Abundance of cheap natural gas from other sources Not applicable Microbial zonation: NRB in the NIWR, SRB deeper in the reservoir Solutions to accelerate implementation Realizing high conversion rates in the eld Not applicable Use of pulsing strategies to reach deep SRB zones Focus on sulde removal in above-ground operations Collaboration of government, industry and academia to gain access Application of state-of-the art sequence technologies to dene corrosive consortia in failed pipe and the effect of treatment

Biocorrosion prevention

Mature/in progress

Lack of access to pipeline spills Uncertainty how to dene corrosion risk Uncertainty how to measure success of treatment strategies May be hard to apply repetitively Pursuit of multiple objectives

MEOR by adding molasses, nutrients. nitrate and ex situ grown strains

Mature/in progress

MEOR by adding nitrate and nutrients only

Mature/in progress

Need more information on success

Consider using native molasses-fermenting population Choose for either oil improvement (no nitrate) or conformance control (excess nitrate) Closely monitor eld applications using this technology in low and high temperature elds.

May not work in high temperature reservoirs

compared to gas may prevent this application from being realized in the short term (Table 1).

Souring prevention with nitrate

Much of the worlds oil is produced by the injection of water to maintain reservoir pressure and sweep the oil to the producing wells. Once injection water breaks through, the fraction of produced water (the water cut) increases with time. Produced water reinjection (PWRI) involves separation of water and oil and mixing produced water with make-up water, drawn from a suitable source, at a water plant (Figure 1). The mixed water is then reinjected. Seawater is injected offshore and in some of the major onshore elds in the Middle East, introducing 28 mM sulfate and causing sulde production by SRB (souring). Including nitrate into seawater has been effective in remediating or preventing souring in elds with resident temperatures above 608C [13], where this can be considered mature technology (Table 1). Nitrate injection induces growth of hNRB and sulde-oxidizing NRB (soNRB). The former compete for the same oil organics as the SRB, whereas the latter oxidize sulde directly. Most soNRB are chemolithotrophs using CO2 as the sole carbon source. SRB are also strongly inhibited by nitrite, produced by NRB. Growth of NRB may be expected downstream from the nitrate injection point and in the near-injection wellbore region (NIWR). Bdtker et al. [14] analyzed backowed injection water from nitrate-amended North Sea reservoirs.
Current Opinion in Biotechnology 2011, 22:15

NR-SOB of the genus Arcobacter and other epsilon-proteobacteria, as well as hNRB of the genus Terasakiella were found. These are mesophiles of which growth is expected in the NIWR, which is cooled down by the seawater injection, but not in the bulk of the reservoir. Switching from injection of 100% fresh seawater to PWRI (75% of hot produced water, 25% cold seawater as make-up water) increased the average temperature of the NIWR and caused more thermophilic SRB and NRB to be isolated [15]. Produced waters from a nitrate-treated eld with a resident temperature of 808C had hNRB of the genus Deferribacter and lower SRB activity at 608C, than in a non-treated eld [16]. No SRB activity was found at 808C. Souring control by nitrate injection into the Enermark eld (Alberta, Canada), which has a low resident temperature (308C) and is injected with water with a low sulfate concentration, decreased produced sulde by 70% in the rst 57 weeks, but this was followed by recovery to prenitrate injection levels [17]. Establishment of discrete zones of hNRB activity in the NIWR and of SRB activity deeper in the reservoir (Figure 1) was hypothesized to cause the recovery. Injected nitrate is unable to inhibit sulde production by SRB under these conditions (Table 1). This problem may not occur in high temperature reservoirs, because deeper regions are too hot for signicant microbial activity. Hence, only the NIWR needs to be nitrate-treated. As a result, nitrate and nitrite breakthrough in producing wells is commonly observed in high but not in low temperature reservoirs.
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Production-related petroleum microbiology: progress and prospects Voordouw 3

Figure 1

WP MW IW I2 I1 water water I3 oil PW

(1)

(2)

(3)
Current Opinion in Biotechnology

Production-related petroleum microbiology in an oil reservoir subjected to produced water reinjection (PWRI). Water injected at injection wells (IW) sweeps an oilwater mixture to the surface at producing wells (PW). Above ground the mixture is separated into produced oil and produced water. The latter enters a water plant (WP), where it is mixed with make-up water (MW) before reinjection in the reservoir. Field-wide control of souring is achieved by injection of nitrate in the WP (I1), displacing sulfate-reducing bacteria (SRB) from (1), the near-injection wellbore region (NIWR). In low temperature reservoirs SRB can grow back deeper in the reservoir (2), complicating the use of nitrate for souring control. Nitrate can also be injected at specific injection wells (I2). Biocides, corrosion inhibitors, sulfide scavengers and other compounds are injected above ground (I1, I3) to protect against corrosion ( ). Microbially enhanced oil recovery (MEOR) can be used to redirect flow at (3) from a path of low resistance (thick arrows) to a productive path of high resistance (dotted arrows) by inducing localized growth of biomass.

Deep SRB activity in low temperature reservoirs can be targeted by injection of high nitrate pulses that exceed the nitrate reduction capacity of NRB in the NIWR. Injection of 760 mM nitrate for 1 hour/week in an injection well from the Enermark eld caused complete removal of sulde from a neighboring production well. Sulde rebounded when the pulsed injections were stopped [17]. Injected nitrate is reduced to nitrite and then to N2 (95%) or ammonium (5%) [18]. The coexistence of ammonium and nitrite induces anammox activity (NH4+ + NO2 ! N2 + 2H2O). Ammonium is also used for the synthesis of increased NRB biomass. These two factors caused ammonium concentrations to decline, especially during periods of increased nitrate injection [18]. The presence of anammox bacteria in the Enermark eld and in an enrichment culture, oxidizing cyclohexane with nitrate [19] was demonstrated. Anammox causes an even greater fraction of injected nitrate to be reduced to N2 at the expense of ammonium.

of 0.3 mM nitrate to a seawater injection system (SWIS) signicantly lowered SRB activity and corrosion rates [20] and compared favorably with biocide treatment. soNRB of the genus Sulfurimonas emerged as major community members in the nitrate-treated SWIS. Microbial communities in the SWIS and in producing wells of an Alaskan North Slope oil eld had Desulfomicrobium thermophilum as the most common SRB [21]. Bioconversion of hydrocarbons to CO2 and acetate may also contribute to corrosion [22]. Hydrogenotrophic SRB or methanogens can use iron as electron donor for sulfate or CO2 reduction causing pitting corrosion. Methanococcus maripaludis strain AK1, isolated from a crude oil storage tank, was very active in this regard [23]. Dening the role of biocorrosion requires rapid access to the most relevant corrosion sites, that is oil pipeline failures. This is unlikely to happen in the current punitive climate associated with spills, causing progress in understanding how microbes contribute to failures to be slower than desirable (Table 1).

Control of souring to control corrosion

Because H2S-rich uids and gases corrode iron, oil and gas companies try to keep sulde concentrations as low as possible to assure pipeline integrity (Figure 1). Addition
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Microbially enhanced oil recovery (MEOR)

Using bacteria either native or introduced to produce more oil is an old idea that is regaining ground primarily because increasing water cuts force companies to adopt
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4 Environmental biotechnology

EOR technologies. MEOR can involve improvement of oil properties (decreasing oil viscosity or interfacial tension) or improvement of reservoir properties by blocking preferred water ow paths (conformance control) or by removing alkane precipitates (waxes). These technologies involve placement of MEOR agents by water injection, cessation of ow to allow reaction and resumption of water injection to produce more oil [24]. Introduction of bacteria is often coupled with introduction of carbohydrate (molasses) as a carbon and energy source. Carbohydrate fermentation typically yields 90% fermentation products (acids, alcohols, gases, biosurfactants) and only 10% biomass offering opportunities to improve oil properties (Table 1). Injecting an ex situ grown strain assures the desired fermentation outcome: in situ production of CO2, acetate, lactate, ethanol, 2,3butanediol and biosurfactant in proportions similar to ex situ fermentations were demonstrated and led to the production of additional oil [25]. However, repeating this strategy may be complicated because a native fermenting population is also likely induced, changing the fermentation outcome of subsequent injections. Relying on native fermenting populations would circumvent this problem. Localized production of biomass and exopolysaccharide is needed for conformance control (Figure 1). Injection of molasses, excess electron acceptor (e.g. nitrate) and nutrients (e.g. phosphate) will give 50% of the injected molasses as biomass with large amounts of CO2 and N2 also being formed. Injection of ex situ grown microorganisms may not be needed. Use of reservoir oil can supplant injection of carbohydrate, making this a simple-to-apply technology (Table 1). Injection of 13 m3 of concentrated nitrate and nutrients, followed by one week cessation of water injection increased oil production by 200% with a 10% decrease in water cut for a year, after which treatment needed to be repeated [26]. Successful application of MEOR strategies requires application of principles of microbial fermentation and growth to the existing reservoir situation. The pursuit of mixed applications to cover all the bases, yielding intermediate amounts of fermentation products and biomass, may seem desirable but the results of these cannot be interpreted and can, therefore, not be improved upon. Making choices requires rigorous understanding of both petroleum and fermentation engineering, a skill set that is not common in todays oil and gas companies.

sequence technologies, allowing routine determination of 107 to 109 bases per sample, will provide more accurate information on who is there and what they are doing, knowledge that is prerequisite to implementing production-related biotechnologies. An outstanding question is how existing and future oil production technologies affect the environment. Injection of biocides, corrosion inhibitors, de-emulsiers, sulde scavengers, oxygen scavengers and other agents is common in todays oil production operations. Use of these compounds is subject to increasingly strict regulations with some biocides being banned outright. PWRI is practiced increasingly even in offshore operations, despite unlimited supply of seawater for injection, to prevent the release of residual oil-containing produced water into the environment. The use of nitrate, which is largely reduced to inert N2, to remove sulde, prevent corrosion or enhance production compares favorably with existing technologies in which more toxic compounds are used. Of course, the grand challenge is to decelerate the slide in EROI, which is the root cause for our mounting environmental problems. Application of new technologies, including petroleum microbiology, will help in this regard.

Acknowledgements
This work was supported by an NSERC Industrial Research Chair Award, which is also supported by Baker Hughes Incorporated, Commercial Microbiology Limited (Intertek), the Computer Modelling Group Limited, ConocoPhillips Company, YPF SA, Aramco Services, Shell Canada Limited, Suncor Energy Developments Inc., and Yara International ASA, as well as by Alberta Innovates-Energy and Environment Solutions. All Industry sponsors and members of the Petroleum Microbiology Research Group are thanked for their efforts and support.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Kaplan JO, Krumhardt KM, Zimmermann N: The prehistoric and preindustrial deforestation of Europe. Quat Sci Rev 2009, 28:3016-3034.

2. 

Hall C, Tharakan P, Hallock J, Cleveland C, Jefferson M: Hydrocarbons and the evolution of human culture. Nature 2003, 426:318-322. A great motivator on why we need to do this. Introduces the concept of EROI. Zengler K, Richnow HH, Rossello-Mora R, Michaelis W, Widdel F: Methaneformation from long-chain alkanes by anaerobic microorganisms. Nature 1999, 401:266-269. First demonstration that anaerobic microbes can make a living converting hexadecane to methane and CO2.

3. 

Microbial communities and environmental considerations

The oil eld community composition has been determined through clone libraries or denaturing gradient gel electrophoresis [14,15,16,19]. Composition changes, for example in response to use of the oxygen scavenger ammonium bisulte, have been documented [27]. New
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4. 

Head IM, Jones DM, Larter S: Biological activity in the deep subsurface and the origin of heavy oil. Nature 2003, 426:344-352. Explains how continued methanogenic microbial oil degradation converts light oil to heavy oil to bitumen. 5. Jones DM, Head IA, Gray ND, Adams JJ, Rowan AK, Aitken CM, Bennett B, Huang H, Brown A, Bowler BFJ et al.: Crude oil biodegradation via methanogenesis in subsurface petroleum reservoirs. Nature 2008, 451:176-180. www.sciencedirect.com

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Production-related petroleum microbiology: progress and prospects Voordouw 5

6.

Gray ND, Sherry A, Larter SR, Erdmann M, Leyris J, Liengen T, Beeder J, Head IM: Biogenic methane production in formation waters from a large gas eld in the North Sea. Extremophiles 2009, 13:511-519.

7. 

Pham VD, Hnatow LL, Zhang S, Fallon RD, Jackson SC, Tomb J-F, DeLong EF, Keeler SJ: Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods. Environ Microbiol 2008, 11:176-187. One of the most detailed analyses of community composition in an oil reservoir. Results by two different methods tended to agree.

18. Cornish-Shartau SL, Yurkiw M, Lin S, Grigoryan AA, Lambo A,  Park H-S, Lomans BP, van der Biezen E, Jetten MSM, Voordouw G: Ammonium concentrations in produced waters from a mesothermic oil eld subjected to nitrate injection decrease through formation of denitrifying biomass and anammox activity 2010 N2. Appl Environ Microbiol 2010, 76:4977-4987. Nitrate injection lowers ammonium concentrations, because anammox causes a larger fraction of injected nitrate to end up as N2. 19. Musat F, Wilkes H, Behrends A, Woebken D, Widdel F: Microbial  nitrate dependent cyclohexane degradation coupled with anaerobic ammonium oxidation. ISME J 2010. [Epub ahead of print]. Clear demonstration of the presence and activity of anammox in a hydrocarbon-fed enrichment culture. These bacteria are likely to be common when nitrate is introduced in oil eld environments. 20. Bdtker G, Thorstenson T, Lilleb B-LP, Thorbjrnsen, Ulven RH,  Sunde E, Torsvik T: The effect of long-term nitrate treatment on SRB activity, corrosion rate and bacterial community composition in offshore water injection systems. J Ind Microbiol Biotechnol 2008, 35:1625-1636. A thorough study on the effect of nitrate on corrosion in a seawater injection system. The news is mostly good for nitrate treatment. 21. Duncan KE, Gieg LM, Parisi VA, Tanner RS, Tringe SG, Bristow J, Suita JM: Biocorrosive thermophilic microbial communities in Alaskan North Slope oil facilities. Environ Sci Technol 2009, 43:7977-7984. 22. Suita JM, Phelps TJ, Little B: Carbon dioxide corrosion and acetate: a hypothesis on the inuence of microorganisms. Corrosion 2008, 64:854-859. 23. Uchiyama T, Ito K, Mori K, Tsurumaru H, Harayama S: Iron corroding methanogen isolated from a crude-oil storage tank. Appl Environ Microbiol 2010, 76:1783-1788. Biocorrosion is not limited to SRB. Methanogens can do it too! 24. Youssef N, Elshahed MS, McInerney MJ: Microbial processes in  oil elds: culprits, problems and opportunities. Adv Appl Microbiol 2009, 66:141-251. A richly laid out review with a focus on MEOR processes. The tables indicate the success or failure of most documented MEOR trials, structures and properties of biosurfactants and much more. 25. Youssef N, Simpson DR, Duncan KE, McInerney MJ, Folmsbee M,  Fincher T, Knapp RM: In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir. Appl Environ Microbiol 2007, 73:1239-1247. The rst and only successful attempt at a complete fermentation balance for carbohydrate injected into the subsurface and metabolized by an injected inoculum. The authors show that biosurfactant produced by this strain is also made downhole. 26. Town K, Sheehy AJ, Govreau BR: MEOR success in southern Saskatchewan. SPE Annual Technical Conference and Exhibition; New Orleans, Louisiana, SPE124319: 2009. 27. van der Kraan GM, Bruining J, Lomans BP, van Loosdrecht MC,  Muyzer G: Microbial diversity of an oil-water processing site and its associated oil eld: the possible role of microorganisms as information carriers from oil-associated environments. FEMS Microbiol Ecol 2010, 71:428-443. Shows a signicant change in community composition (more SRB) upon application of ammonium bisulte, universally used as oxygen scavenger in the treatment of oil eld waters.

8. McInerney MJ, Sieber JS, Gunsalus RP: Syntrophy in anaerobic  global carbon cycles. Curr Opin Biotechnol 2009, 30:623-632. Syntrophs are at the heart of methanogenic oil transformation. Their physiology, biochemistry and molecular biology are explained in detail. 9.  Gieg LM, Davidova IA, Duncan KE, Suita JM: Methanogenesis, sulfate reduction and crude oil biodegradation in hot Alaskan oilelds. Environ Microbiol 2010. [Epub ahead of print]. Indicates which oil components are most rapidly converted to methane. 10. Siddique T, Fedorak PM, MacKinnon MD, Foght JM: Metabolism  of BTEX and naphtha compounds to methane in oil sands tailings. Environ Sci Technol 2007, 41:2350-2356. Substantial methane is formed in tailing ponds from methanogenic conversion of naphta diluent. The bitumen itself is believed to be inert. 11. Kaster KM, Voordouw G: Effect of nitrite on a thermophilic, methanogenic consortium from an oil storage tank. Appl Microbiol Biotechnol 2006, 72:1308-1315. 12. Gieg LM, Duncan KE, Suita JM: Bioenergy production via  microbial conversion of residual oil to natural gas. Appl Environ Microbiol 2008, 74:3022-3029. Indicates the extent to which methane from oil could contribute to our energy supply. 13. Sunde E, Torsvik T: Microbial control of hydrogen sulde  production in oil reservoirs. In Petroleum Microbiology. Edited by Olliver B, Magot M. Washington, DC: ASM Press; 2005:201213. Demonstrates that injection of low nitrate concentrations removes souring from high temperature elds. 14. Bdtker G, Lysnes K, Torsvik T, Bjrnestad E, Sunde E:  Microbial analysis of backowed injection water from a nitrate-treated North Sea oil reservoir. J Ind Microbiol Biotechnol 2009, 36:439-450. Gives a good indication of what needs to be done to learn what inhabits the NIWR. The best would be to get core, but that is not possible. 15. Lysnes K, Bdtker G, Torsvik T, Bjrnestad E, Sunde E: Microbial response to reinjection of produced water in an oil reservoir. Appl Microbiol Biotechnol 2009, 83:1143-1157. 16. Gittel A, Sorensen KB, Skovhus TL, Ingvorsen K, Schramm A: Prokaryotic community structure and sulfate reducer activity in water from high-temperature oil reservoirs with and without nitrate treatment. Appl Environ Microbiol 2009, 75:7086-7096. 17. Voordouw G, Grigoryan AA, Lambo A, Lin S, Park H-S, Jack TR,  Coombe D, Clay B, Zhang F, Ertmoed R et al.: Sulde remediation by pulsed injection of nitrate into a low temperature Canadian heavy oil reservoir. Environ Sci Technol 2008, 43:9512-9518. It is rare that so much information is made available on an existing eld. A good example of what collaboration between academia and industry can bring.

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