Margarita L. Miroshnichenko

Radioisotopic, Culture-Based, and Oligonucleotide Microchip Analyses of Thermophilic Microbial Communities in a Continental High-Temperature Petroleum Reservoir

ABSTRACT Activity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H2S liter−1 day−1 occurred at 60 and 80°C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH4 liter−1 day−1. Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the genera Thermotoga, Thermoanaerobacter, Geobacillus, Petrotoga, Thermosipho, and Thermococcus, the latter four being represented by new species. Except for Thermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of the Aquificales/Desulfurobacterium-Thermovibrio cluster and of the genera Desulfurococcus and Thermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.

Dissimilatory reduction of Fe(III) by thermophilic bacteria and archaea in deep subsurface petroleum reservoirs of western siberia

Abstract. Twenty-five samples of stratal fluids obtained from a high-temperature (60–84°C) deep subsurface (1700–2500 m) petroleum reservoir of Western Siberia were investigated for the presence of dissimilatory Fe(III)-reducing microorganisms. Of the samples, 44% and 76% were positive for Fe(III) reduction with peptone and H2 respectively as electron donors. In most of these samples, the numbers of culturable thermophilic H2-utilizing iron reducers were in the order of 10–100 cells/ml. Nine strains of thermophilic anaerobic bacteria and archaea isolated from petroleum reservoirs were tested for their ability to reduce Fe(III). Eight strains belonging to the genera Thermoanaerobacter, Thermotoga, and Thermococcus were found capable of dissimilatory Fe(III) reduction, with peptone or H2 as electron donor and amorphous Fe(III) oxide as electron acceptor. These results demonstrated that Fe(III) reduction may be a common feature shared by a wide range of anaerobic thermophiles and hyperthermophiles in deep subsurface petroleum reservoirs.

Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent

A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13 degrees N). When grown under elevated hydrostatic pressure, the cells are rod-shaped with a sheath-like outer structure, motile, have a mean length of 1-1.5 microm and stain Gram-negative. They appear singly or in short chains. When grown at lower, or atmospheric, pressures, the cells elongate and become twisted. Growth is enhanced by hydrostatic pressure; the optimal pressure for growth is 40 MPa (26 MPa pressure at sampling site). The temperature range for growth is 45-70 degrees C, the optimum being around 65 degrees C (doubling time is approximately 20 min at 40 MPa). Growth is observed from pH 5 to pH 8, the optimum being at pH 6. The salinity range for growth is 10-50 g NaCl l(-1), the optimum being at 30 g l(-1). The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur. The G+C content of the genomic DNA is 29 +/- 1 mol%. According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain. On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as the type species. The type strain is KA3T (= DSM 14283T = JCM 11233T).

Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).

Recent developments in the thermophilic microbiology of deep-sea hydrothermal vents

The diversity of thermophilic prokaryotes inhabiting deep-sea hot vents was actively studied over the last two decades. The ever growing interest is reflected in the exponentially increasing number of novel thermophilic genera described. The goal of this paper is to survey the progress in this field made in the years 2000–2005. In this period, representatives of several new taxa of hyperthermophilic archaea were obtained from deep-sea environments. Two of these isolates had phenotypic features new for this group of organisms: the presence of an outer cell membrane (the genus Ignicoccus) and the ability to grow anaerobically with acetate and ferric iron (the genus Geoglobus). Also, our knowledge on the diversity of thermophilic bacteria from deep-sea thermal environments extended significantly. The new bacterial isolates represented diverse bacterial divisions: the phylum Aquificae, the subclass Epsilonproteobacteria, the order Thermotogales, the families Thermodesulfobacteriaceae, Deferribacteraceae, and Thermaceae, and a novel bacterial phylum represented by the genus Caldithrix. Most of these isolates are obligate or facultative lithotrophs, oxidizing molecular hydrogen in the course of different types of anaerobic respiration or microaerobic growth. The existence and significant ecological role of some of new bacterial thermophilic isolates was initially established by molecular methods.

Metabolic Versatility and Indigenous Origin of the Archaeon Thermococcus sibiricus, Isolated from a Siberian Oil Reservoir, as Revealed by Genome Analysis

ABSTRACT Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a “saccharolytic gene island” absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and β-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.

Thermococcus gorgonarius sp. nov. and Thermococcus pacificus sp. nov.: heterotrophic extremely thermophilic archaea from New Zealand submarine hot vents

Two extremely thermophilic archaea, designated W-12 and P-4, were isolated from a geothermal vent in the tidal zone of Whale Island, New Zealand, and from geothermally heated bottom deposits of the Bay of Plenty, New Zealand, respectively. Cells of isolate W-12 are irregular cocci, 0.3-1.2 microns in diameter, motile with polar flagella. The cell envelope consists of one layer of subunits with a major protein of M(r) 75,000. Cells produce protrusions of different kinds: prostheca-like, chains of bubbles, or network of fimbriae. Cells of isolate P-4 are regular cocci, 0.7-1.0 micron in diameter, motile with polar flagella. The cell envelope consists of two layers of subunits; its major protein has an M(r) of 56,000. Both organisms are obligate anaerobes, fermenting peptides in the case of strain W-12, or peptides and starch in the case of P-4. Elemental sulfur is required for growth and is reduced to hydrogen sulfide. The optimal growth temperature of the new isolates is in the range 80-88 degrees C. The optimal growth pH is 6.5-7.2. The G + C content of the DNA of strain W-12 is 50.6 mol%, and of strain P-4 is 53.3 mol%. Based on physiological characteristics, 165 rDNA sequence comparison and DNA base composition, the new isolates were considered to be members of the genus Thermococcus. The low level of DNA-DNA hybridization with the type strains of other Thermococcus species confirms the novel species status of the new isolates. The new isolates are described as Thermococcus gorgonarius sp. nov., with type strain W-12 (= DSM 10395T), and Thermococcus pacificus sp. nov., with type strain P-4 (= DSM 10394T).

Thermoproteus uzoniensis sp. nov., a new extremely thermophilic archaebacterium from Kamchatka continental hot springs

A new extremely thermophilic rod-shaped archaebacterium was found in the samples from hot springs and soil of the Uzon caldera (SW of Kamchatka pen.). Cells are rods from 1 to 20 μm in length and 0.3 to 0.4 μm in width, sometimes branching or with spherical protrusions on the ends. The cell wall consists of two layers: an internal one with distinct hexagonal structure and the outer one with less clear structure and variable thickness. Cells are non-motile and have no flagella. The new organism grows anaerobically by fermenting peptides, concurrently reducing elemental sulfur to H2S. Fermentation products are acetate, isobutyrate, isovalerate. The G+C content of the DNA is 56.5 mol. %. A new species Thermoproteus uzoniensis is described. Type strain is isolate Z-605, DSM 5262.

Caldicellulosiruptor kronotskyensis sp. nov. and Caldicellulosiruptor hydrothermalis sp. nov., two extremely thermophilic, cellulolytic, anaerobic bacteria from Kamchatka thermal springs

Five novel strains (2002(T), 2902, 2006, 108(T) and 117) of cellulose-degrading, anaerobic, thermophilic bacteria were isolated from terrestrial hot springs of Kamchatka (Far East, Russia). Strains 2002(T) and 108(T) were non-spore-forming bacteria with a Gram-positive type cell wall and peritrichous flagella. Optimum growth of strains 2002(T) and 108(T) occurred at pH 7.0 and at temperatures of 70 and 65 degrees C, respectively. The G+C contents of the DNA of strains 2002(T) and 108(T) were 35.1 and 36.4 mol%, respectively. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Caldicellulosiruptor. However, DNA-DNA hybridization experiments indicated that the levels of relatedness between strains 2002(T) and 108(T) and those of recognized members of the genus Caldicellulosiruptor ranged between 32 and 54 %. Based on both phenotypic and genomic differences, strains 2002(T) and 108(T) are considered to represent two novel species of the genus Caldicellulosiruptor. The names proposed for these organisms are Caldicellulosiruptor kronotskyensis sp. nov. (type strain 2002(T)=DSM 18902(T)=VKM B-2412(T)) and Caldicellulosiruptor hydrothermalis sp. nov. (type strain 108(T)=DSM 18901(T)=VKM B-2411(T)).

Petrotoga olearia sp. nov. and Petrotoga sibirica sp. nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia.

Strictly anaerobic, thermophilic bacteria (strains SL24T, SL25T, SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia). These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales. On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25T, SL27, SL29 and SL32 were identical. Therefore, only strains SL24T and SL25T were studied in detail. The optimum temperature for growth of both strains was 55 degrees C. Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1). The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C contents of the genomic DNA of strains SL24T and SL25T were respectively 35 and 33 mol%. Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences. Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24T = DSM 13574T = JCM 11234T) and Petrotoga sibirica (type strain SL25T= DSM 13575T = JCM 11235T) are proposed.