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Microbial Ecology of Oil Fields

The role of hyperthermophilic in oil fields

Karl O. Stetter , Robert Huber

Lehrstuhl für Mikrobiologie, University of Regensburg, 93053 Regensburg, Germany

ABSTRACT

Deep geothermally heated oil stratifications represent newly recognized non-volcanic islands of microbial life at extremely high temperatures, some 3,500 m below the surface. Samples of produced fluids contain high concentrations of various anaerobic extremely thermophilic and hyperthermophilic and bacteria, indicating the presence of complex microbial communities in situ. The isolates consist of heterotrophs and facultative and obligate chemolithoautotrophs, some of which are able to use crude oil in their energy- yielding reactions. They belong to the genera , , , Methanococcus, Methanobacterium, Thermotoga, Thermosipho, and Thermodesulfobacterium. Most isolates are members of already known from hot springs and hydrothermal vents. They may have entered the reservoirs through natural routes such as faults and oil seeps or by sea water injection. A great deal are sulfidogens and may, therefore, participate in reservoir "souring" at temperatures previously considered too high for biochemical reactions. Introduction Microbial formation in shallow oil reservoirs (reservoir "souring") at moderate temperatures is well known and is mainly caused by a variety of mesophilic and slightly thermophilic sulfate-reducing bacteria [12]. Deep hot oil reservoirs, however - in view of their extremely high temperatures around 100°C - were previously considered as too extreme to support microbial activity and reservoir "souring" observed there was attributed exclusively to abiotic chemical processes [5]. During the last decade, hyperthermophilic archaea and bacteria which grow at temperatures of 80 to 113°C were isolated from terrestrial hot springs and submarine hot vents [10, 11]. By their physiological properties and 16S rRNA sequence, represent a variety of very different taxonomic groups. Within the 16S rRNA-based of life, hyperthermophiles cover all the deepest and shortest branches [8, 11]. Archaeoglobus is a unique hyperthermophilic sulfate reducing archaeon. Its first isolation from a submarine hot vent close to Island () led to the hypthesis that "souring" of hot reservoirs may be caused biotically by Archaeoglobus-like organisms [7]. In this paper, we describe the isolation, properties and possible role of extreme and hyperthermophiles, which we have discovered for the first time in deep hot oil reservoirs [9].

Microbial Biosystems: New Frontiers Proceedings of the 8th International Symposium on Microbial Ecology Bell CR, Brylinsky M, Johnson-Green P (ed) Atlantic Canada Society for Microbial Ecology, Halifax, Canada, 1999. Microbial Ecology of Oil Fields

Results

Sampling, Isolation, and Identification In order to investigate the presence of extreme thermophiles and hyperthermophiles in deep oil-bearing strata, anaerobic samples (each 100 ml) from the upcoming reservoir fluids were taken from 15 wells at the Thistle offshore oil production platform (East Shetland Basin, North Sea) and from 33 wells at the Prudhoe Bay, Endicott and Kuparuk oil fields (North Slope of Alaska, U.S.A.). The maximum temperatures measured in the reservoirs were between 71 and 110°C. At the wellheads, the produced fluids had cooled to between 20°C and 87°C, depending on the flow rate (up to 3.5 m/sec). The produced fluids consisted of oil, water and gas in varying ratios. The water cut varied between 10% and 30%, depending on the sample source. The separated, depressurized water phase of the samples had a pH close to 7 with an ionic strength similar to seawater. All four reservoirs were continuously flooded by pressurized seawater and had a history of H2S production. Hyperthermophiles were detected after inoculating sterile anaerobic culture media (containing various possible substrates) with the water phases of the samples and incubating them at 85 and 102°C. Original cell concentrations were determined by serial dilution of the water phases. The species were identified by DNA/DNA hybridization with the type species already known from hydrothermal systems.

Table 1. Examples of viable hyperthermophiles found in Thistle produced fluids (Enrichments at 85 and 102°C) Well No. Original titre Identified species (cells/ml) A02 100 Archaeoglobus fulgidus; Archaeoglobus lithotrophicus 10 Thermococcus celer; 1000 Pyrococcus sp. nov. A05 1000 Archaeoglobus fulgidus; Archaeoglobus lithotrophicus 10 Thermococcus litoralis 10 Pyrococcus sp. nov. A08 10000 Archaeoglobus fulgidus; A. profundus; A. lithotrophicus 100 Thermococcus litoralis 10 Pyrococcus sp. nov. A25 100 Archaeoglobus fulgidus; Archaeoglobus lithotrophicus 1000 Thermococcus celer; Thermococcus litoralis 10 Pyrococcus sp. nov. A31 100 Archaeoglobus fulgidus; Archaeoglobus lithotrophicus 10 Thermococcus celer; Thermococcus litoralis 1000 Pyrococcus sp. nov.

The samples from the Thistle reservoir were available first and were studied in more detail. Our studies revealed that all 15 Thistle samples contained mixtures of hyperthermophiles. The original cell titres (in the water phases) were 10 to 10,000 viable cells/ml (Table 1), indicating very high cell concentrations in the reservoirs. The Thistle hyperthermophiles identified consist of members of Archaeoglobus fulgidus, Archaeoglobus Microbial Ecology of Oil Fields lithotrophicus, Archaeoglobus profundus, Thermococcus celer and Thermococcus litoralis. These archaeal species had been found before in submarine hydrothermal systems [11]. In addition, a new species of Pyrococcus was detected in the Thistle samples (Table 1). Enrichments at 65°C gave rise to cultures of Methanococcus thermolithotrophicus (5 samples), Methanobacterium thermoautotrophicum (2 samples) and a new species of Thermosipho (1 sample; Table 2).

Table 2. Maximum growth temperature and morphology of hot reservoir thermophiles

Species Tmax (°C) Morphology

Bacteria

Thermosipho sp. 78 rods in chains with sheath, overballooning ends Thermotoga sp. 90 rods with sheath, overballooning ends Thermodesulfobacterium sp. 80 rods

Archaea

Methanobacterium thermoautotrophicum 70 rods in chains, UV-fluorescent Methanococcus thermolithotrophicus 70 irregular cocci, UV-fluorescent, ∅ 1.5µm

Archaeoglobus lithotrophicus 89 Archaeoglobus profundus 90 irregular cocci, often triangular, ∅ 0.5 - 2µm Archaeoglobus fulgidus 94 UV-fluorescent like

Thermococcus celer 93 Thermococcus litoralis 98 irregular cocci, often in pairs, Pyrococcus sp. 103 ∅ 2µm

From about 50% of the samples taken from the Alaskan oil reservoirs, Archaeoglobus and Thermococcus-like cultures could be enriched at 85°C. Enrichment cultures from 7 samples contained Thermotoga-like organisms. Depending on the samples, the original cell concentrations (viable cells/ml) ranged from 1 to 10 for Archaeoglobus, 1 to 10,000 for Thermococcus, and 1 to 1,000 for Thermotoga. Cultures of rod-shaped bacteria resembling members of the Thermodesulfobacterium, were obtained at 75°C from 2 samples. Microbial Ecology of Oil Fields

Physiological properties In the laboratory, the deep reservoir isolates grow at temperatures corresponding to the in situ conditions (Table 3; Fig. 1). Archaeoglobus lithotrophicus grows between 63 and 89°C with an optimum around 80°C (optimal doubling time: 95 min; Fig. 1).

Investigation of the pressure tolerance of Archaeoglobus fulgidus demonstrated its ability to grow between 300 and 420 atmospheres under reservoir conditions (not shown). In their energy-yielding reactions, a great deal of the reservoir thermophiles are sulfidogens (Table 3). Members of Archaeoglobus are sulfate reducers. As electron donor, Archaeoglobus fulgidus is able to use a variety of laboratory substrates [7]. In contrast, Archaeoglobus lithotrophicus is a strict chemolithoautotroph. Members of Pyrococcus and Thermococcus are able to reduce elemental using complex organic material as electron donor (Table 3). 0 In the absence of S , members of these genera are able to grow by with H2, CO2, and fatty acids as end products. Thermosipho and Thermotoga ferment , 0 forming H2, CO2, acetate and lactic acid. In the presence of S , however, H2S is formed instead of H2 [3]. Methanobacterium thermoautotrophicum and Methanococcus thermolithotrophicus are obligate chemolithoautotrophs forming methane from H2 and CO2. Microbial Ecology of Oil Fields

Table 3. Growth temperature and laboratory substrates of isolates from the Thistle reservoir Species Growth temperature (°C) Laboratory substrates Electron min. opt. max. (positive requirement) acceptor

2- Archaeoglobus fulgidus 60 83 94 lactate; cell extract SO4 2- Archaeoglobus 65 82 90 H2/CO2 + acetate SO4 profundus 2- Archaeoglobus 63 80 89 H2CO2 SO4 lithotrophicus sp. nov. Thermococcus litoralis 50 88 98 cell extract; peptone S0 Thermococcus celer 75 87 93 cell extract; peptone S0 Pyrococcus sp. Nov. 60 92 103 cell extract; peptone S0

Pure cultures of the strains were obtained by plating on to culture medium solidified by 0.6% gelrite (Kelco, San Diego, USA). 10 ml cultures were grown during shaking at 200 rpm in stoppered 120 ml serum bottles. Organic substrates were added at 0.05 % (w/v). Cell extract consisted of extract (Difco), meat extract (Difco) and Methanothermus fervidus extract. Alternative substrates for growth are given after the semicolons.

In the presence of S0, however, these methanogens are able to form high concentrations of H2S [6]. Thermodesulfobacterium is a facultative chemolithoautotrophic sulfate reducer which grows with H2 or lactate as electron donors. Interaction with crude oil So far, nothing is known about the natural substrates of the hyperthermophiles in the hot oil reservoirs. Initial experiments with Archaeoglobus fulgidus demonstrate, that this organism is able to use an unidentified component of Thistle crude oil as electron donor, most likely a fatty acid (Table 4).

Table 4. Archaeoglobus fulgidus: heterotrophic anaerobic growth on oil components by 2- SO4 -reduction Substrate Growth Crude oil (Thistle) + Crude oil, water-extracted + Crude oil, alcali water-extracted - Olive oil + Arachnic acid +

In addition, this organism is able to grow on olive oil and on arachnic acid. Recently, total sequencing revealed that Archaeoglobus fulgidus possesses 57 genes encoding enzymes in ß-oxidation of fatty acids [4]. In a further screening, various species of sulfur-reducing hyperthermophiles were examined for their potential to desulfurize crude Microbial Ecology of Oil Fields oils (Ex Champion Crude, 0.13% sulfur; Arabian Light Crude, 2.1% sulfur; Tia Juana Pesado Crude, 2.8% sulfur; all topped at 150°C). Sulfur was omitted from the culture media and was substituted by 20% (v/v) of crude oil or 5% (v/v) of gasoline. Surprisingly, members of Thermococcus, Pyrococcus and Thermotoga were active in desulfurization, as indicated by H2S formation. For example, approximately 50% desulfurization of the Ex Champion Crude oil was obtained with Thermococcus VC 20 (85°C; 48 hours; oil: seawater mixture 20:80), whereas Thermococcus VC 16 and Thermotoga PB 12 were able to remove sulfur (15 and 10%, respectively) from the Tia Juana Pesado crude oil. Both strains of Thermococcus had been isolated from submarine hot vents, while Thermotoga was an isolate from the Prudhoe Bay hot oil reservoir. Independently of their ability to form H2S, in the presence of crude oil, several hyperthermophiles (e.g. 7 different isolates of Thermotoga) exhibited up to ten times higher final cell densities. Concomitantly, the physical properties of the oils changed by the emulsifying activities of the organisms. Conclusions Our findings demonstrate that deep oil reservoirs are a novel geothermally heated environment for extremely thermophilic and hyperthermophilic archaea and bacteria. Since these organisms were recovered from 4 separate oil fields they are likely to be widely distributed in hot, seawater-flooded petroleum fields [9]. Very recently, our results have been confirmed for oil reservoirs in France and Japan [1, Takahata pers. comm.]. Due to their high growth temperatures, pressure tolerance and highly anaerobic mode of life, the hot reservoir organisms appear to be well adapted to a subterranean environment of heated porous rocks, saturated with petroleum fluids and water. All of our hot reservoir isolates are able to reduce sulfur compounds which may be provided in situ by reservoir minerals, crude oil and injected seawater. In addition, unknown components of crude oil serve as electron donors in their energy-yielding anaerobic respiration type metabolism. The isolates may be a critical part of a complex reservoir food web. Concerning the origin of the hot reservoir organisms, there is no evidence and it appears unlikely that isolated ("autochthonous") communities may have survived since the filling of the reservoirs: temperatures of several hundred degrees (e.g. 300 - 500°C) and extremely high pressures during their geologic history should have prevented this. On the other hand, there is compelling evidence that the reservoirs may have been infected much later (a) via natural routes such as faults and oil seeps, and (b) by flooding of reservoirs with seawater which contains viable hyperthermophiles in low population densities [2, 9]. Also, the practice of discharging produced water is likely to increase the local titre of hyperthermophiles in oil producing areas of the North Sea, thus increasing the probability of inoculating other deep, hot reservoirs. In the Alaskan fields, where hyperthermophiles-laden produced water is reinjected into the formation, the continuous reinoculation of the reservoirs is inevitable. Thus our findings present evidence for the existence of deep subterranean pockets of microbial life several kilometers below the surface. References 1. Haridon S L', Reysenbach A-L, Glénat P, Prieur D, Jeanthon C (1995) Hot subterranean biosphere in a continental oil reservoir. Nature 377:223-224 Microbial Ecology of Oil Fields

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