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Keywords:

  • Methanoplanaceae;
  • Methanoplanus;
  • Methanogen;
  • Taxonomy;
  • Oil well

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

A disc-shaped methanogenic bacterium designated strain SEBR 4847T (T=type strain) was isolated from a sample collected from an African offshore oil field. Strain SEBR 4847T was non-motile, had a G+C content of 50 mol% and produced methane from H2+CO2, formate, and CO2+propanol. Strain SEBR 4847T grew optimally at 37°C; no growth was observed at 25°C or 45°C. It grew in the presence of up to 50 g/l NaCl; 10–30 g/l was required for optimal growth. The optimum pH for growth was 7.0. Doubling time was about 10 h under optimal conditions. Based on 16S rRNA sequence analysis, the isolate was identified as a new species of the genus Methanoplanus and designated Methanoplanus petrolearius sp. nov. The type strain is SEBR 4847T (=OCM 486).


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The study of the microbiology of oil reservoirs has shown the presence of fermentative bacteria [10, 19], sulfate reducers [6, 22, 20, 24], acetogens [9], and methanogens [3, 8, 15–18]. The methanogens that were isolated and characterized include (i) the hydrogenotrophic Methanobacterium thermoautrophicum[15], M. bryantii[8], Methanococcus thermolithotrophicus[16], and Methanobacterium ivanovii[3], (ii) phenotypic variants of Methanobacterium thermoaggregans[15], and M. thermoalcaliphilum[8], (iii) the methylotrophic Methanococcoides euhalobius[17], and (iv) the acetoclastic Methanosarcina mazei[18].

We have recently undertaken extensive microbial studies of oil fields. We report here on the enumeration, isolation and characterization of a new species of a dominant hydrogenotrophic methanogen present in an offshore oil field of the Gulf of Guinea, West Africa.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Sample collection and sample source

One liter sample was collected from the well-head of an offshore oil field of the Gulf of Guinea, West Africa, as previously described [4]. The in situ temperature was 33°C and the NaCl concentration was 32 g/l. The samples were air mailed to our laboratory and stored at 4°C until used.

2.2Enumeration, enrichment, isolation and growth conditions

Enrichment, enumeration, and isolation of methanogenic cultures were achieved in a basal medium that contained (per liter) 1 g of NH4Cl, 0.3 g of K2HPO4, 0.3 g of KH2PO4, 5 g of MgCl2, 2 g of CaCl2·2H2O, 0.2 g of KCl, 30 g of NaCl, 0.5 g of CH3COONa, 0.5 g of cysteine-HCl, 1 g of yeast extract (Difco Laboratories, Detroit, MI), 1 g of bio-Trypticase (bioMérieux, France), 10 ml of the trace mineral element solution of Balch et al. [2], 1 mg of resazurin, and 1000 ml of distilled water. The pH was adjusted to 7.0 with 10 M KOH. The medium was boiled under a stream of O2-free N2 gas, cooled to room temperature and 5 ml and 20 ml aliquots dispensed under a stream of N2/CO2 (80:20) gas mixture into Hungate tubes and serum bottles, respectively, and autoclaved for 45 min at 110°C. Na2S·9H2O and Na2CO3 were injected from sterile stock solutions to a final concentration of 0.04% and 0.2% prior to culture inoculations.

Enumeration of methanogenic bacteria was performed using the Most Probable Number (MPN) technique. Tubes containing basal medium were amended with H2/CO2 (80:20, 2 bars) or a mixture of methanol (40 mM) and acetate (20 mM) as growth substrates and were inoculated in triplicate with serial dilutions prepared from the oil-field sample. Results were recorded after incubation at 37°C for 7 days by measuring methane production. Enrichment cultures were initiated by inoculating 1 ml of the last dilution of the positive hydrogenotrophic methanogen enumeration tubes, into serum bottles containing basal medium and H2/CO2 (80:20, 2 bars). The inoculated serum bottles were incubated at 37°C without shaking. Pure cultures were obtained by the repeated use of the Hungate roll tube method [12] using growth medium solidified with 1.5% Noble agar (Difco).

2.3pH, temperature, and NaCl ranges for growth

For pH studies we used Hungate tubes containing the growth medium, the pH of which was adjusted to the desired value by injecting appropriate volumes of sterile 10% NaHCO3 or Na2CO3 anaerobic stock solutions. Growth was tested at temperatures ranging from 25°C to 45°C. To determine salt requirement for growth, NaCl was weighed directly into Hungate tubes and the medium was subsequently dispensed as described above. The strain was subcultured at least once under the same experimental conditions prior to inoculation.

2.4Substrate utilization

Substrates were added from sterile stock solutions to the basal medium at a final concentration of 10 mM (acetate, trimethylamine, lactate, glucose, 1-propanol, 2-propanol, 1-butanol, isobutanol) or 40 mM (formate, methanol). Hydrogen oxidation was tested using H2/CO2 (80:20, 2 bars) in the gas phase.

2.5Analytical techniques

Unless otherwise indicated, all experiments were performed in duplicate. Phase contrast and fluorescence microscopy were performed as previously described [5]. Growth was quantified by inserting tubes directly into a model UV-160A spectrophotometer (Shimadzu Corp., Kyoto, Japan) and measuring the optical density at 580 nm. Methane was measured as described previously [7].

2.6Determination of G+C content

The G+C content of DNA was determined at DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. The DNA was isolated and purified by chromatography on hydroxyapatite, and the G+C content was determined by using high-performance liquid chromatography (HPLC) as described by Mesbah et al. [14]. Nonmethylated lambda DNA (Sigma) was used as the standard.

2.716S rRNA sequence studies

A primer pair, designated FARCH-9 (5′-CTGGTTGATCCTGCCAG-3′) and Rd1 (5′-AAGGAGGTGATCCAGCC-3′) was used to amplify the 16S rRNA gene from genomic DNA of the methanogen. The amplified product was purified [1] and the sequence determined with an ABI automated DNA sequencer by using a Prism dideoxy terminator cycle sequencing kit and the protocol recommended by the manufacturer (Applied Biosystems Inc.). The primers used for sequencing were F2 (5′-CAGGATTAGATACCCTGGTAG-3′), R2 (5′-GTATTACCGCGGCTGCTG-3′), R4 (5′-CCGTCAATTCCTTTGAGTTT-3′) and the two amplification primers designated FARCH9 and Rd1 described above.

The 16S rRNA gene sequence which we determined was manually aligned with reference sequences of various members of the domain Archaea by using the alignment editor `ae2' [13]. Reference sequences were obtained from the Ribosomal Database Project [13]. Positions of sequence and alignment uncertainty were omitted from the analysis. A pairwise evolutionary distances based on 1217 unambiguous nucleotides was computed by using the method of Jukes and Cantor and dendrograms were constructed from these distances by using the neighbour-joining method. Both programmes form part of the PHYLIP package [11].

2.8Nucleotide sequence accession number

The 16S rRNA gene sequence of strain SEBR 4847T has been deposited in the Genbank database under accession number U76631.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Bacterial enumeration

MPN estimations indicated that 1.9×103 cells/ml hydrogenotrophic methanogens and 5×102 cells/ml methylotrophic non-acetoclatic methanogens were present in the oil-field sample. Total microflora, estimated by microscopy under epifluorescence, was 4.5×103 cells/ml.

3.2Enrichment and isolation

After 1 week of incubation at 37°C, positive enrichment cultures developed in tubes that had been inoculated from the last dilution of positive hydrogenotrophic methanogen enumeration tubes. Microscopic examination revealed the presence of disc-shaped bacteria. Circular colonies developed in agar roll tubes after 1 month incubation at 37°C. Two axenic cultures were obtained using this technique. They appeared similar in morphology and physiology; therefore only strain SEBR 4847T (T=type strain) was further characterized and designated the type strain.

3.3Morphology

Strain SEBR 4847T was a strictly anaerobic, non-motile, irregular disc-shaped bacterium with a diameter of 1–3 μm, occurring singly or in pairs (Fig. 1).

image

Figure 1. Phase-contrast micrograph of strain SEBR 4847T showing irregular disc shaped cells (DS). Bar=5 μm.

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3.4Optimum growth conditions

Strain SEBR 4847T grew at an optimum temperature between 35 and 40°C. It did not grow at 25°C or at 45°C. The isolate grew in the presence of NaCl concentrations ranging from 0 to 5%, with an optimum between 1 and 3% NaCl. Growth occurred between pH 5.3 and pH 8.4 with an optimum at pH 7.0.

3.5Substrate used for growth

Strain SEBR 4847T used H2–CO2, formate, and CO2+2-propanol to produce methane. Strain SEBR 4847T could not utilize acetate, methanol, trimethylamine, lactate, glucose, CO2+1-propanol, CO2+1-butanol, and isobutanol. Acetate was required for growth, and the presence of yeast extract was stimulatory for growth.

3.6G+C content of DNA

The G+C content of isolate SEBR 4847T was 50 mol%.

3.716S rRNA gene sequencing and sequence analysis

Using five primers, we determined an almost complete sequence consisting of 1429 bases of the 16S rRNA gene of strain SEBR 4847T. Phylogenetic analysis revealed that strain SEBR 4847T was a member of the family Methanoplanaceae, order Methanomicrobiales and the closest relatives was Methanoplanus limicola and Methanoplanus endosymbiosus (average similarity of 93.5%). A dendrogram generated by the Neighbour-Joining method showing this relationship is shown in Fig. 2. Bootstrap analysis indicated that the relationship of strain SEBR 4847T to M. limicola and M. endosymbiosus was not robust.

image

Figure 2. Dendrogram showing the position of strain SEBR 4847T amongst the methanogenic bacteria. Bar indicates evolutionary distance.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Strain SEBR 4847T produced methane from H2 and CO2 and a green fluorescence under UV light microscope, thus indicating that the isolate was a methanogen. Strain SEBR 4847Tis a disc-shaped irregular bacterium and has a G+C content of 50 mol% and therefore cannot be placed as member of the order Methanococcales which are irregular cocci and have G+C content ranging from 30 to 40 mol%. The order Methanomicrobiales contains families Methanomicrobiaceae, Methanocorpusculaceae, Methanoplanaceae, and Methanosarcineae. Acetate, methanol or methylamines cannot be used as substrates; therefore strain SEBR 4847T cannot be placed as member of the family Methanosarcinaceae. Members of the families Methanomicrobiaceae and Methanocorpusculaceae are irregular cocci or rods; they are therefore morphologically distinct from strain SEBR 4847T. Methanoplanus which is the only described genus in the family Methanoplanaceae, is comprised of two species, namely Methanoplanus endosymbiosus and M. limicola. Both are described as disc-shaped hydrogenotrophic methanogens [23, 25] and therefore resemble strain SEBR 4847T morphologically and phenotypically. The analysis of the 16S rRNA sequence of strain SEBR 4847T confirmed its affiliation to M. limicola and M. endosymbiosus (average similarity 93.5%). Strain SEBR 4847T differed from M. endosymbiosus[23] in the temperature growth range and a lower G+C content in the DNA (Table 1). In contrast to M. limicola[25], strain SEBR 4847T grew at 42°C and had an optimum NaCl concentration ranging from 1% up to 3%. Phylogenetically, M. limicola and strain SEBR 4847T were also distinct (similarity of 95%), a feature which alone warrants to place SEBR 4847T as a new species of the genus Methanoplanus[21].

Table 1.  Characteristics that differentiate members of the genus Methanoplanus
SpeciesStrain SEBR 4847TaMethanoplanus limicolabMethanoplanus endosymbiosusc
  1. aThis study. bData from Wildgruber et al. [25]. cData from van Bruggen et al. [23]. ND=not determined.

Type strainOCM 486DSM 2279DSM 3599
SourceAfrican oil wellswampmarine ciliate
Temp range (°C)28–4317–4116–36
Optimum Temp (°C)374032
pH range5.3–8.2ND6.1–8.0
Optimum pH7.06.5–7.56.8–7.3
NaCl concn. range (%)0–50.4–5.40–4.5
Optimum NaCl concn. (%)1–311.5
Generation time (h)1077
G+C content (mol %)504839
Substrates usedH2+CO2,H2+CO2,H2+CO2,
 formateformateformate
 CO2+2-propanol  

Strain SEBR 4847T was isolated from a subsurface ecosystem. In contrast, M. limicola was isolated from a small Italian swamp containing drilling waste and was assumed to inhabit swamps of freshwater and seawater. However, on the basis of our results we can hypothesize that methanogens similar to M. limicola and strain SEBR 4847T could originate from subsurface ecosystems since phylogenetically similar strains were also isolated from another offshore oil field off the Gulf of Guinea (data not shown). Furthermore strain SEBR 4847T exhibited optimum growth over a wide range of NaCl concentration (1–3%) indicating that it could grow optimally in the saline conditions of the oil field (3%). Based on phylogenetic and phenotypic characteristics, we propose that strain SEBR 4847T be designated as a new species of the genus Methanoplanus, M. petrolearius sp. nov.

4.1Description of Methanoplanus petrolearius sp. nov.

Methanoplanus petrolearius (pe.tro.le.a'riusL. fem. n. petra rock; L. adj. olearius related to vegetal oil; petrolearius L. masc. adj. related to mineral oil). Round colonies (diameter: 1–2 mm) are present after 3 weeks of incubation at 37°C. Cells are irregular disc-shaped with a diameter of 1–3 μm. The cells occur singly or in pairs and are non-motile under microscope. Methanogenic and obligately anaerobic member of the domain Archaea. The optimum temperature for growth is 37°C with no growth occurring at 25°C and 45°C. The optimum pH is 7.0; growth occurs from pH 5.3 to 8.4. The optimum NaCl concentration for growth is between 1 and 3% NaCl with growth occurring at NaCl concentration ranging from 0 and 5%. Doubling time is about 10 h under optimal conditions. Produces methane from H2–CO2, formate, and CO2+2-propanol. The strain requires acetate for growth and yeast extract is stimulatory. The strain cannot use acetate, methanol, trimethylamine, lactate, glucose, CO2+1-propanol, CO2+1-butanol, and isobutanol. The G+C content of the DNA is 50% (as determined by HPLC). Isolated from oil-producing well. The type strain is SEBR 4847T(=OCM 486).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Funding in part from the Australian Research Council to B.K.C.P. and from Elf Aquitaine to J.L.C. is gratefully acknowledged.

References

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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