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1 IRD, UR 101 Extrêmophiles, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL, Marseille, France
2 School of Biomolecular and Biomedical Sciences, Griffith University, Brisbane, Australia
Correspondence
Bernard Ollivier
ollivier{at}esil.univ-mrs.fr
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ABSTRACT |
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-Proteobacteria. As strain 4BONT is physiologically and phylogenetically different from H. thermoluteolus, it is proposed that it be assigned to a novel species of a novel genus, Petrobacter succinatimandens gen. nov., sp. nov. The type strain is 4BONT (=DSM 15512T=CIP 107790T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Petrobacter succinatimandens 4BONT is AY219713.
A transmission electron micrograph of strain 4BONT and a graph showing the effect of temperature on the growth of strain 4BONT cultivated on succinate are available from IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Among these micro-organisms, heterotrophic together with hydrogenotrophic micro-organisms (e.g. methanogens and sulfate-reducing bacteria) are considered as common inhabitants of oilfield environments (Ivanov et al., 1983; Fardeau et al., 1993, 1997, 2000; Grassia et al., 1996; Magot et al., 2000; Mueller & Nielsen, 1996; Nilsen et al., 1996; Ollivier et al., 1998). Despite the fact that organic acids are known to occur in oil reservoirs (Rees et al., 1997), a great deal of attention has been paid by microbiologists to heterotrophic micro-organisms using carbohydrates in these environments (Fardeau et al., 1993, 1997, 2000; Magot et al., 2000); therefore, little information is available on the isolation of heterotrophic non-sulfate-reducing bacteria that use organic acids (e.g. fumarate, succinate, etc.) aerobically or anaerobically. The only organic-acid-using micro-organisms isolated from oilfield ecosystems, unable to use sulfate as terminal electron acceptor, include members of the genera Marinobacter (Huu et al., 1999), Deferribacter (Greene et al., 1997), Denitrovibrio (Myhr & Torsvik, 2000) and Anaerobaculum (Rees et al., 1997).
Here, we describe a novel moderately thermophilic, organic-acid-using, nitrate-reducing bacterium (strain 4BONT), belonging to the -Proteobacteria, isolated from a non-water-flooded Australian terrestrial oil reservoir. It presented significant phenotypic, genotypic and phylogenetic differences when compared with members of the -Proteobacteria, which led to the proposal of it being assigned to a novel genus, Petrobacter, as the type and sole species, Petrobacter succinatimandens.
The oil sample used in this study was collected from the Riverslea oilfield in the Bowen–Surat basin of Eastern Australia (Queensland). The sample was designated OCA5 and was stored at 4 °C until used. Enrichment was performed in a medium prepared anaerobically (Fardeau et al., 2000) containing (l–1 distilled water) 0·3 g K2HPO4, 0·3 g KH2PO4, 0·2 g MgCl2.6H2O, 0·1 g CaCl2.2H2O, 0·1 g KCl, 1 g NaCl, 1 g NH4Cl, 10 mM CH3COONa, 1 g yeast extract (Difco Laboratories) and 10 ml of the trace mineral solution of Balch et al. (1979). The pH was adjusted to 7·0 with 10 M KOH. The vessels were autoclaved for 45 min at 110 °C and, prior to inoculation, 10 mM KNO3 and 0·5 % (v/v) of the vitamin solution of Balch et al. (1979) were added from sterile stock solutions. A H2/CO2 mixture (2 bars) was added in the gas phase. For enrichment, a 2 ml oil-well water sample was inoculated into 20 ml of medium and incubated at 50 °C without agitation. Three enrichment series were performed in the same medium before isolation. Strains were isolated by repeated use of the Hungate roll-tube technique (Hungate, 1969), with medium solidified with 2 % agar noble (Difco). The basal medium, used for characterization of pH, temperature and NaCl ranges for growth of the isolates, was similar to the enrichment medium supplemented with 20 mM sodium succinate. The culture medium was adjusted to different pH values by injecting NaHCO3 from 10 % (w/v) sterile anaerobic stock solutions. For studies on NaCl requirements, NaCl was weighed directly in the tubes prior to dispensing the medium. Substrates were tested in aerobiosis (nitrogen was replaced by air in the gas phase) and anaerobiosis (in the presence and in the absence of nitrate as terminal electron acceptor) in basal medium at a final concentration of 20 mM. As a test for electron acceptors, sodium thiosulfate (20 mM), sodium sulfate (20 mM), sodium sulfite (2 mM), elemental sulfur (1 %, w/v), potassium nitrate (10 mM) and potassium nitrite (2 mM) were added to the medium. The use of electron acceptors was evaluated by measuring the optical density at 580 nm and by measuring H2S, ammonium, nitrite or nitrous oxide production.
Sulfide was determined photometrically as colloidal CuS by using the method of Cord-Ruwisch (1985). Nitrite was detected by the colorimetric method of Griess–Illosvay (Knapp & Clark, 1984) and by the Quantofix test (Macherey-Nagel). Nitrous oxide was measured by gas chromatography (Fardeau et al., 1993). Organic compounds were determined as described previously (Fardeau et al., 1997). Morphological characteristics of isolates were observed with an optiphot (Nikon) phase microscope. Electron microscopy studies were performed as described by Koussémon et al. (2001).
The G+C content of the DNA of strain 4BONT was determined at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) by using HPLC as described by Mesbah et al. (1989). Non-methylated lambda DNA (Sigma) was used as the standard. The 16S rRNA gene of the isolate was amplified as described previously (Miranda-Tello et al., 2003). The PCR products were purified by using a NucleoSpin Extract kit (Macherey Nagel) and were cloned into the vector PGEM-T-easy (Promega). Plasmids containing the insert were purified using the Wizard Plus SV Minipreps DNA Purification System (Promega), according to the manufacturer's protocol. Sequencing was carried out by Genome Express (Grenoble, France). The new sequence was aligned to a full-length 16S rDNA consensus sequence, assembled and checked manually for accuracy using the alignment editor BIOEDIT v5.0.9 (Hall, 1999). The 16S rRNA gene sequence of strain 4BONT was compared with other sequences in the GenBank database (Benson et al., 1999) and RDP (Maidak et al., 2001), using BLAST (Altschul et al., 1997) to identify its closest relatives. The sequences of the closest relatives were retrieved and, together with the 16S rRNA gene sequence of strain 4BONT, were subjected to a phylogenetic analysis. Positions of sequence and alignment ambiguity were omitted from the analysis and the pair-wise evolutionary distances based on 1374 unambiguous nucleotides were computed by using the method of Jukes & Cantor (1969). A dendrogram was constructed using the neighbour-joining method (Saitou & Nei, 1987). Confidence in the tree topology was determined by using 100 bootstrapped trees (Felsenstein, 1993). The accession numbers for the 16S rDNA sequences of the reference organisms are included in Fig. 1.
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The in situ physicochemical conditions, together with the presence of phenotypically and phylogenetically closely related anaerobes in oilfields, suggest that oil reservoirs are mainly anaerobic ecosystems (Magot et al., 2000). However, the accidental introduction of oxygen into oil reservoirs cannot be excluded due to water-flooding during oil extraction. This might partly explain the occurrence of aerobic to microaerophilic bacteria in oilfield ecosystems (Gevertz et al., 2000; Voordouw et al., 1996; Telang et al., 1997). Among these micro-organisms, hydrogen-oxidizers, using nitrate as their terminal electron acceptor, have been isolated and characterized (Gevertz et al., 2000). Attempts to isolate phenotypically similar micro-organisms from the Australian oil reservoirs that we studied were unsuccessful. Despite the presence of hydrogen as energy source in the enrichment medium that we used, our microbiological studies only led to the isolation of heterotrophic bacteria that used organic acids, aerobically or anaerobically, but not hydrogen. Isolation of these bacteria was only allowed by the presence of yeast extract in the original enrichment medium. The subsequent isolation on other substrates of organic-acid-users originating from oilfield ecosystems has already been reported (Davydova-Charakhch'yan et al., 1992; Rees et al., 1997). Strain 4BONT, isolated from an Australian oil reservoir in Queensland, was further characterized and was shown to oxidize a limited range of organic acids (pyruvate, fumarate, succinate) together with ethanol. Despite the fact that strain 4BONT is an aerobic, nitrate-reducing bacterium, it differed phylogenetically from strains CVO and FWKO B, isolated from Canadian oilfields, as they both belong to the epsilon subdivision of the Proteobacteria. In contrast, strain 4BONT belongs to the beta subdivision of the Proteobacteria, as indicated by the phylogenetic tree shown in Fig. 1
, and it oxidizes neither elemental sulfur nor thiosulfate. Strain 4BONT also differed significantly from other nitrate-reducing bacteria isolated from oil reservoirs: Deferribacter thermophilus is a strict anaerobe which is most closely related to Flexistipes sinusarabici (Greene et al., 1997); Denitrovibrio acetiphilus is a strict anaerobe which oxidizes acetate and has Geovibrio ferrireducens as its closest phylogenetic relative (Myhr & Torsvik, 2000); and Marinobacter aquaeolei is a facultative, mesophilic acetate-oxidizer (Huu et al., 1999). Comparison of the almost-complete 16S rDNA sequence of strain 4BONT (1529 nt) with sequences in the public databases indicated that the strain was related to a group of uncultured micro-organisms (LaPara et al., 2000) (mean similarity of 98 %; data not shown). Nevertheless, the bacterial species phylogenetically most closely related to strain 4BONT is Hydrogenophilus thermoluteolus (Hayashi et al., 1999) (sequence similarity of 91·8 %). Strain 4BONT differs from H. thermoluteolus by its inability to oxidize hydrogen and by having a lower DNA G+C content (58·6 mol% for strain 4BONT vs 63–65 mol% for H. thermoluteolus). Because of its distinct phenotypic, genotypic and phylogenetic characteristics, it is proposed that strain 4BONT represents a novel species, Petrobacter succinatimandens, within a novel genus, Petrobacter, within the -Proteobacteria.
Description of Petrobacter gen. nov.
Petrobacter (Pe.tro.bac'ter. Gr. fem. n. petra rock, stone; M.L. masc. n. bacter equivalent of Gr. neut. n. bakterion rod, staff; N.L. masc. n. Petrobacter the stone bacterium).
Cells are straight rods. Gram-negative. Spores are not formed. Grow aerobically and anaerobically only with nitrate. Moderately thermophilic member of the domain Bacteria, class -Proteobacteria. Organic acids serve as main substrates.
The type species is Petrobacter succinatimandens.
Description of Petrobacter succinatimandens sp. nov.
Petrobacter succinatimandens (suc.ci.na.ti.man'dens. N.L. n. succinatum succinate; L. part. adj. mandens eating, consuming; N.L. masc. adj. succinatimandens consuming succinate).
Cells are straight rods (0·3–0·4x2 µm), which occur singly or in pairs and possess one polar flagellum. Spores are not formed. Stains Gram-negative. Round colonies (1–2 mm diameter) develop in roll tubes after 1 week incubation at 50 °C. Chemo-organotroph. Obligate aerobe. Oxidase- and catalase-positive. Moderately thermophilic. The optimum temperature for growth is 55 °C at pH 7; temperature range between 35 and 60 °C. The optimum pH for growth is 6·9; growth occurs between pH 5·5 and 8·0. Slightly halotolerant, growing in the presence of up to 3 % NaCl, with an optimum at 0·5 %. Oxidizes fumarate, pyruvate, succinate, ethanol and yeast extract in the presence of oxygen or nitrate as terminal electron acceptor. The following compounds are not used in aerobiosis or in the presence of nitrate as terminal electron acceptor: acetate, butyrate, propionate, valerate, isovalerate, lactate, adipate, alanine, L-glutamate, proline, serine, valine, fructose, glucose, lactose, maltose, ribose, xylose, vanillate, benzoate, 4-hydroxybenzoate, 3-methylbenzoate, phenylacetate, 4-aminobenzoate, benzaldehyde. Nitrate is reduced to nitrous oxide. Elemental sulfur, sulfate, thiosulfate and nitrite are not used as electron acceptors. Isolated from an Australian oil well.
The type strain is 4BONT (=DSM 15512T=CIP 107790T). The G+C content of the DNA is 58·6 mol% (HPLC).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Balch, W. E., Fox, G. E., Magrum, R. J., Woese, C. R. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol Rev 43, 260–296.
Benson, D. A., Boguski, M. S., Lipman, D. J., Ostell, J., Ouellette, B. F. F., Rapp, B. A. & Wheeler, D. L. (1999). GenBank. Nucleic Acids Res 27, 12–17.
Cord-Ruwisch, R. (1985). A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate-reducing bacteria. J Microbiol Methods 4, 33–36.
Davydova-Charakhch'yan, I. A., Mileeva, A. N., Mityushina, L. L. & Belyaev, S. S. (1992). Acetogenic bacteria from oil fields of Tataria and western Siberia. Microbiology (English translation of Mikrobiologiya) 61, 208–216.
Fardeau, M.-L., Cayol, J.-L., Magot, M. & Ollivier, B. (1993). H2 oxidation in the presence of thiosulfate by a Thermoanaerobacter strain isolated from an oil-producing well. FEMS Microbiol Lett 113, 327–332.[CrossRef]
Fardeau, M.-L., Ollivier, B., Patel, B. K. C., Magot, M., Thomas, P., Rimbault, A., Rocchiccioli, F. & Garcia, J.-L. (1997). Thermotoga hypogea sp. nov., a xylanolytic, thermophilic bacterium from an oil-producing well. Int J Syst Bacteriol 47, 1013–1019.
Fardeau, M.-L., Magot, M., Patel, B. K. C., Thomas, P., Garcia, J.-L. & Ollivier, B. (2000). Thermoanaerobacter subterraneus sp. nov., a novel thermophile isolated from oilfield water. Int J Syst Evol Microbiol 50, 2141–2149.[Abstract]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.
Gevertz, D., Telang, A. J., Voordouw, G. & Jenneman, G. E. (2000). Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine. Appl Environ Microbiol 66, 2491–2501.
Grassia, G. S., McLean, K. M., Glénat, P., Bauld, J. & Sheehy, A. J. (1996). A systematic survey for thermophilic fermentative bacteria and archaea in high temperature petroleum reservoirs. FEMS Microbiol Ecol 21, 47–58.
Greene, A. C., Patel, B. K. C. & Sheehy, A. J. (1997). Deferribacter thermophilus gen. nov., sp. nov., a novel thermophilic manganese- and iron-reducing bacterium isolated from a petroleum reservoir. Int J Syst Bacteriol 47, 505–509.
Hall, T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hayashi, N. R., Ishida, T., Yokota, A., Kodama, T. & Igarashi, Y. (1999). Hydrogenophilus thermoluteolus gen. nov., sp. nov., a thermophilic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium. Int J Syst Bacteriol 49, 783–786.
Hungate, R. E. (1969). A roll-tube method for the cultivation of strict anaerobes. Methods Microbiol 3B, 117–132.
Huu, N. B., Denner, E. B. M., Ha, D. T. C., Wanner, G. & Stan-Lotter, H. (1999). Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well. Int J Syst Bacteriol 49, 367–375.
Ivanov, M. V., Belayaev, S. S., Zyakun, A. M., Bondars, V. & Laurinivicius, K. (1983). Microbiological methane formation in oil field development. Geokhimiya 11, 1647–1654.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Knapp, J. S. & Clark, V. L. (1984). Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect Immun 46, 176–181.
Koussémon, M., Combet-Blanc, Y., Patel, B. K. C., Cayol, J.-L., Thomas, P., Garcia, J.-L. & Ollivier, B. (2001). Propionibacterium microaerophilum sp. nov., a microaerophilic bacterium isolated from olive mill wastewater. Int J Syst Evol Microbiol 51, 1373–1382.[Abstract]
LaPara, T. M., Nakatsu, C. H., Pantea, L. & Alleman, J. E. (2000). Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl Environ Microbiol 66, 3951–3959.
Magot, M., Ollivier, B. & Patel, B. K. C. (2000). Microbiology of petroleum reservoirs. Antonie van Leeuwenhoek 77, 103–116.[CrossRef][Medline]
Maidak, B. L., Cole, J. R., Lilburn, T. G. & 7 other authors (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29, 173–174.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Miranda-Tello, E., Fardeau, M. L., Fernandez, L., Ramirez, F., Cayol, J.-L., Thomas, P., Garcia, J.-L. & Ollivier, B. (2003). Desulfovibrio capillatus sp. nov., a novel sulfate-reducing bacterium isolated from an oil field separator located in the Gulf of Mexico. Anaerobe 9, 97–103.
Mueller, R. F. & Nielsen, P. H. (1996). Characterization of thermophilic consortia from two souring oil reservoirs. Appl Environ Microbiol 62, 3083–3087.[Abstract]
Myhr, S. & Torsvik, T. (2000). Denitrovibrio acetiphilus, a novel genus and species of dissimilatory nitrate-reducing bacterium isolated from an oil reservoir model column. Int J Syst Evol Microbiol 50, 1611–1619.[Abstract]
Nilsen, R. K., Beeder, J., Thorstenson, T. & Torsvik, T. (1996). Distribution of thermophilic marine sulfate reducers in North Sea oil field waters and oil reservoirs. Appl Environ Microbiol 62, 1793–1798.[Abstract]
Ollivier, B., Fardeau, M.-L., Cayol, J.-L., Magot, M., Patel, B. K. C., Prensier, G. & Garcia, J.-L. (1998). Methanocalculus halotolerans gen. nov., sp. nov., isolated from an oil-producing well. Int J Syst Bacteriol 48, 821–828.
Rees, G. N., Patel, B. K. C., Grassia, G. S. & Sheehy, A. J. (1997). Anaerobaculum thermoterrenum gen. nov., sp. nov., a novel, thermophilic bacterium which ferments citrate. Int J Syst Bacteriol 47, 150–154.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Telang, A. J., Ebert, S., Foght, J. M., Westlake, D. W. S., Jenneman, G. E., Gevertz, D. & Voordouw, G. (1997). Effect of nitrate injection on the microbial community in an oil field as monitored by reverse sample genome probing. Appl Environ Microbiol 63, 1785–1793.[Abstract]
Voordouw, G., Armstrong, S. M., Reimer, M. F., Fouts, B., Telang, A. J., Shen, Y. & Gevertz, D. (1996). Characterization of 16S rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria. Appl Environ Microbiol 62, 1623–1629.[Abstract]
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