Microbial Ecology of Oil Fields The role of hyperthermophilic prokaryotes 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 archaea 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 Archaeoglobus, Thermococcus, Pyrococcus, Methanococcus, Methanobacterium, Thermotoga, Thermosipho, and Thermodesulfobacterium. Most isolates are members of species 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 hydrogen sulfide 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, hyperthermophiles represent a variety of very different taxonomic groups. Within the 16S rRNA-based phylogenetic tree 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 Vulcano Island (Italy) 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 thermophiles 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; Thermococcus litoralis 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 methanogens 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 genus 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 sulfur using complex organic material as electron donor (Table 3). 0 In the absence of S , members of these genera are able to grow by fermentation with H2, CO2, and fatty acids as end products. Thermosipho and Thermotoga ferment carbohydrates, 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 yeast extract (Difco), meat extract (Difco) and Methanothermus fervidus extract. Alternative substrates