MICROBIOLOGY LETTERS ELSEVIER FEMS Microbiology Letters 147 (1997) 51-56

kfetlzanoplc~mspetrolenrius sp. nov., a novel methanogenic bacterium from an oil-producing well

B. Ollivier >', J.-L. Cayo1 B.K.C. Pate1 ').'k7 M. Magot c, M.-L. Fardeau J.-L. Garcia ''

Abstract

A disc-shaped methanogenic bacterium designated stlain SEBR 1S47T (T= type strain) was isolated from a sample collected from an African offshore oil field. Strain SEBR 4S47= was non-motile. had a Gi-C content of 50 mol'% and produced methane from Hz+CO.. formate. and COs+propanol. Strain SEBR 4S4.i' grew optimally at 37°C: no growth was observed at 35°C or 45°C. It grew in the presence of up to 50 dl NaCl: 10-30 g/l was required for optimal growth. The optimum pH for growth was 7.0. IO was Doubling time was about h under optimal conditions. Based on 16s rRXA sequence :malysis, the isolate identified as a new of the gentis .i/rrhrr~i~~~~~i/i~i/.~Lind designated .~~erh~~~zupkr~ztrsper~ul~i~~izi.ssp. nov. The type strain is SEBR 4547' (= OCM 356').

1. Introduction phenotypic variants of .~l~~rli~iiiohti~.te~iziiizr/ierinoag- ,ore,v(iiis [ 151, and Jl. tlzerr7?oalc,irlipililllllZ[SI, (iii) the The study of the microbiology of oil reservoirs has methylotrophic .Cf~tk~iiiococcoi~iseuhalobizrs [lÍ], shown the presence of Fermentative bacteria [IC), I?], and (iv) the acetoclasric .C~~~I~L~I~~SIII.~~~~CImrrei [ 181. sulfate reducers [6.11.30,34], acetogens [?I, and We havz recently undertaken extensive microbial methanogens [3.S. IS-lS]. The methanogens that studies of oil fields. We report here on the enumer- were isolated and characterized include (i) the hydro- ation. isolation and characterization of a new species genotrophic i~fetlrm7obacrerilii72 theririocizitropliicim~ of a dominant hydrogenotrophic merhanogen [ 151, M.hryanrii [SI, IC~ec/rciizococni.sthern~olirlzotl-o- present in an offshore oil field of the Gulf of Guinea, phis [ 161, and ~lferlianobcicrerizii7zivcmovii [3], (ii) West Africa.

* Curresponding author. Tel.: (7) 3S75 t6l (7) +61 7695: fax: 3S75 7656: e-mail: b.patel(~sct.gu.edu.au

by 037S-IO97/97/S17.O0 Copyright C 1997 Published Elsevier Science B.V. All rights reserved 8 O 6 10 -0 PII SO 3 7 - 1 9 7 I9 O 50 I 2. Ahterials and methods spectively, and autoclaved for 45 min at 110°C. Na2S.9H20 and Nn.?CO:j were injected from sterile stock solutions to a final concentration of 0.04'%1and 0.20/0 prior to culture inoculations. One liter sample was collected from the well-head Enumeration of methanogenic bacteria was per- of an offshore oil field of the Gulf of Guinea, West formed using the h4ost Probable Number (MPN) Africa, as previously described [4]. The in situ tem- technique. Tubes containing basal medium were perature was 33°C and the NaCl concentration was amended with Hz/CO:! (S0:70,2 bars) or a mixture 32 g/l. The samples were air mailed to our laboratory of methanol (40 mM) and acetate (20 mM) as and stored at 4°C until used. growth substrates and were inoculated in triplicate with serial dilutions prepared from the oil-field sam- ple. Results were recorded after incubation nt 37°C for 7 days by measuring methane production. En- richment cultures were initiated by inoculating 1 ml Enrichment. enumeration, and isolation of metli- of the last dilution of the positive hydrogenotrophic anogenic cultures were achieved in a basal medium methanogen enumeration tubes. into serum bottles that contained [per liter) 1 g of NH.,CI, 0.3 g of containing basal medium and H2/CO1 (50:70, 2 KzHPO,, 0.3 g of KH2POL,5 g of MgCl?, 7 g of bars). The inoculated serum bottles were incubated g CaCIy?H20. 0.7 g of KCI. 30 g of NnCI, 0.5 of at 37°C without shaking. Pure cultures were ob- 0.5 p of cysteine-HC1. 1 g of yeast ex- tained by the repeated Lise of the Hungate roll tube CH:iCOONa. MI). tract (Difco Laboratories. Detroit. 1 g of bio- method [ 121 using growth medium solidified with Trypticase ibiohl&-ieux. France). 10 ml of the trace 1.5% Noble agar (Difco). mineral element solution of Balch et al. ['l. I mg of resazurin. and 1000 ml of distilled water. The pH M wx adjusted to 7.0 with 10 KOH. The medium was boiled under a stream of 02-free N? gas, cooled For pH studies we used Hungate tubes containing to room temperature and 5 ml and 70 ml aliquots the growth medium. the pH of which was adjusted to dispensed under a stream of N2ICO2 (S0:70) gas the desired value by injecting appropriate volumes of mixture into Hungate tubes and serum bottles, re- sterile 10% NaHC0:j or NazCO:: anaerobic stock

Table 1 Chnmctcrisrics that dirferentiure members of the .~J~~//~~/~u~i~~/i~/~

Species Strain SEBR 4847'.' .~frririiriop/nriirsIiiuicoiu" .\Icrllil~lopill/lll.s I c.rrtlo.syiii h ic~.str.s' 486 Type strain OC41 DSM 7179 DSM 3599 Source African oil well swamp marine ciliate 1 Temp ranze ("Cl 25-43 1741 16-36 :q Optimum Temp ("C) 37 '40 <- pH range 5.3-8.2 ND 6.1-8.0 Optimum pH 7.0 6.5-7.5 6.3-7.3 NnCl concn. range (?

'This study. "Data from Wildgruber et 31. [25]. "Data from van Bruggen et al. [3]. cil. jl-56 B. Ollivier et I FEIMS i~~icrobiolo~yLeriers 147 (1997) 53 solutions. Growth was tested at temperatures rang- sequencer by using a Prism dideoxy terminator cycle ing from 3°C to 45°C. To determine salt require- sequencing kit and the protocol recommended by the ment for growth, NaCl was weighed directly into manufacturer (Applied Biosystems Inc.). The primers Hungate tubes and the medium was subsequently used for sequencing were F2 (5’-CAGGATTAGA- dispensed as described above. The strain was subcul- TACCCTGGTAG-3’), R3 (5‘-GTATTACCGCGG- tured at least once under the same experimental con- CTGCTG-3 ‘), R4 (5 ’-CC GTCAATTCCTTTGAG- ditions prior to inoculation. TTT-3’) and the two amplification primers designated FARCH9 and Rdl described above. -7.4. Substrate utilizotiori The 16s rRNA gene sequence which we deter- mined was manually aligned with reference sequen- Substrates were added from sterile stock solutions ces of various members of the domain by to the basal medium at a final concentration of 10 using the alignment editor ‘ae2’ [l3]. Reference se- mM (acetate, trimethylamine, lactate, glucose, 1- quences were obtained from the Ribosomal Data- propanol, 2-propanol. 1-butanol, isobutanol) or 10 base Project [13]. Positions of sequence and align- mM (formate, methanol). Hydrogen oxidation was ment uncertainty were omitted from the analysis. A tested using H2/C02 (80:10,2 bars) in the gas phase. pairwise evolutionary distances based on 121 7 unam- biguous nucleotides was computed by using the method of Jukes and Cantor and dendro,urams were constructed from these distances by using the Unless otherwise indicated, all experiments were neighbourjoining method. Both programmes form performed in duplicate. Phase contrast and fluores- part of the PHYLIP package [II]. cence microscopy were performed as previously de- scribed [5]. Growth was quantified by inserting tubes 2.8. Nucleotide sequerice iiccessiori iiiitnber directly into a model UV-1 60A spectrophotometer (Shimadzu Corp., Kyoto: Japan) and measuring The 16s rRNA gene sequence of strain SEBR the optical density at 580 nm. Methane was meas- 4547* has been deposited in the Genbank database ured as described previously [7]. under accession number U7663 1.

-7.6.Deterininatiori of G+ C content 3. Results The G+C content of DNA was determined at DSM-Deutsche Sammlung von Mikroorganismen 3.1. Bacterid eiiiinieratiot7 und Zellkulturen GmbH, Braunschweig, Germany. The DNA was isolated and purified by chromatog- MPN estimations indicated that 1.9 X lo3 cells/ml X raphy on hydroxyapatite, and the G+C content was hydrogenotrophic methanogens and 5 10’ cellslml determined by using high-performance liquid chro- methylotrophic non-acetoclatic methanogens were matography (HPLC) as described by Mesbah et al. present in the oil-fieid sample. Total microflora, esri- [IA]. Nonmethylated lambda DNA (Sigma) was used mated by microscopy under epifluorescence, was x as the standard. 4.5 IO3 cells/ml.

2.7. 16s rRiVA sequence studies 3.2. Eiirichmen t arid isolotiori

A primer pair, designated FARCH-9 (5’-CTGGT- After 1 week of incubation at 37C, positive en- TGATCCTGCCAG-3’) and Rdl (5’-XAGGAGGT- richment cultures developed in tubes that had been GATCCXGCC-3’) was used to amplify the 16s inoculated from the last dilution of positive hydro- rRNA gene from genomic DNA of the methanogen. genotrophic merhanogen enumeration tubes. Micro- The amplified product was purified [l] and the se- scopic examination revealed the presence of disc- quence determined with an ABI automated DNA shaped bacteria. Circular colonies developed in I47 (1997) 51 B. Ollivier et ul. IFEMS Microbinlogv Letters 51-56

or at 45°C. The isolate grew in the presence of NaCl concentrations ranging from O to 5%t with an opti- mum between 1 and 3% NaCl. Growth occurred between pH 5.3 and pH 8.4 with an optimum at pH 7.0.

3.5. Substrate trsedfor growth

Strain SEBR 4847' used Hz-COz, formate, and CO?+Z!-propanoI to produce methane. Strain SEBR 4847T could not utilize acetate, methanol, trimethyl- OP Fig. 1. Phase-contrast micrograph strain SEBR 4S47T showing amine, lactate, glucose, CO?+l-propanoi, CO?+l- irregular disc shaped cells (DS). Bar = 5 pin. butanol, and isobutanol. Acetate was required for growth, and the presence of yeast extract was stim- ulatory for growth. agar roll tubes after 1 month incubation at 37°C. oj Two axenic cultures were obtained using this techni- 3.6. G+C coiitent DNA que. They appeared similar in morphology and phys- iology; therefore only strain SEBR 4847T (T = type The G+C content of isolate SEBR 4847T was 50 strain) was further characterized and designated the mol%,. type strain. analysis 3.7. 16s rRXA geize seqiiencìng and sequence

Using five primers, we determined an almost com- Strain SEBR 4S47T was a strictly anaerobic, non- plere sequence consisting of 1429 bases of the 16s motile, irregular disc-shaped bacterium with a diam- rRNA gene of strain SEBR 4SJ7T. Phylogenetic eter of 1-3 um, occurring singly or in pairs (Fig. 1). analysis revealed that strain SEBR 4847T was a member of the family Methanoplanaceae, order 3. J. Opriininn gioivtlz corirlitiotis and the closest relatives was i~~~thcitioplnrziisliinicola and 2tfethrinoplciniisenrlos.vnz- Strain SEBR grew at an optimum temper- biosiis (average similarity of 93.5%). A dendrogram ature between 35 and 40°C. It did not grow at 25°C generated by the Neighbour-Joining method showing

IOII .I.letIianoplaiius endosyrnbiosiis c ,lJerlianoplanzis Iirnicolu Llfet/ianoplanuspetroleariiis

.Ifer/ianocorpiisciiblrnparviim Metlianosaera thermoacetoplzila Metethannsarcinu thennophilu ,Eleletlianokalopliilus mahii .I.~etIianotlienniisfervidiis ikfeletlianobacteriumrherinoaritoh.ophiczim ~bfetlianococcusjannaschii Metlianococciis vannielii

0.10

Fig. 2. Dendrogram showing the position of strain SEBR 4847T amongst the methanogenic bacteria. Bar indicates evolutionary distance. 147 (1997) B. 0llisic.r et u¡. I FEicIS Microbiology Letters 51-55 ' 55 this relationship is shown in Fig. 2. Bootstrap anal- was assumed to inhabit swamps of freshwater and ysis indicated that the relationship of strain SEBR seawater. However, on the basis of our results we M. 4547T to il% liiiiicoln and eizclos~~mhiosuswas not can hypothesize that methanogens similar to ibf. hi- robust. colci and strain SEBR 4S47T could originate from subsurface' ecosystems since phylogenetically similar strains were also isolated from another otyshore oil 4. Discussion field off the Gulf of Guinea (data not shown). Fur- thermore strain SEBR 4S47T exhibited optimum Strain SEBR 4547T produced methane from H2 growth over a wide range of NaCl concentration and CO? and a green fluorescence under UV light (1-3%) indicating that it could grow optimally in microscope. thus indicating that the isolate was a the saline conditions of the oil field (3'%1).Based on methanogen. Strain SEBR ?S47Tis a disc-shaped ir- phylogenetic and phenotypic characteristics. we pro- regular bacterium and has -a G+C content of 50 pose that strain SEBR 4S47r be designated as a new mol%, and therefore cannot be placed 'as member species of the genus i~fethciiioplriizus,M. petro1carirr.r of the order Methanococcales which are irregular sp. nov. cocci and have G+C content ranging from 30 to 40 mol%. The order ~lethaIioniicrobi¿iIescontains fam- ilies ilIethanomicrobiaceae, ~lethanocorpusculaceae. Methanoplanaceae, and ~lethanosarcineae.Acetate. methanol or methylamines cannot be used as sub- Methmoplniz~uyetroleariia (pe.tro.le.a'riusL. fem. strates; therefore strain SEBR 4S47T cannot be n. petra rock; L. adj. olecíriiis related to vegetal oil: placed as member of the family ~rethanosarcinaceae. prrrolearizw L. masc. adj. related to mineral oil). Members of the families and Round colonies (diameter: 1-3- mm) are present aftzr hlethanocorpusculaceae are irregular cocci or rods ; 3 weeks of incubation at 37°C. Cells are irregular the]: are therefore morphologically distinct from disc-shaped with a diameter of 1-3 pm. The cells strain SEBR 4S47T. il.1erhaizoplnizzis which is the occur singly or in pairs and are non-motile under only described genus in the family Methanoplana- microscope. Methanogenic and obligately anaerobic ceae. is comprised of two species. namely :bfethciizo- member of the domain Archaea. The optimum tem- phiizís eiiclosj~iirbiosus and ,I% linricolcl. Both are perature for growth is 37°C with no growth occur- described as disc-shaped hydrogenotrophic methano- ring at 15°C and 45°C. The optimum pH is 7.0; gens [23?15] and therefore resemble strain SEBR growth occLirs from pH 5.3 to 5.4. The optimum 4S47r morphologically and phenotypically. The NaCl concentration for growth is between 1 and analysis of the 16s rRYA sequence of strain SEBR 3OA NaCl with growth occurring at NaCl concentra- 4S47T confirmed its affiliation to M. liinicoln and M. tion ranging from O and 5%. Doubling time is about eiztios~iirbiosio (average similarity 93.5%). Strain 10 h under optimal conditions. Produces methane SEBR 4S47T differed from Jf. enc/osyiizbioszís [Y] from H?-CO?, formate, and CO?+'-propanol. The in the temperature growth range and a lower'G+C strain requires acetate for growth and yeast extract is content in the DNA (Table 1). In contrast to M. stimulatory. The strain cannot use acetate, methanol. limicolsi [35], strain SEBR 4S47' grew at 12°C and trimethylamine, lactate, glucose. CO.?+l-propanol. had an optimum YaCl concentration ranging from CO?+l-butanol, and isobutanol. The G+C content 1'% up to 34:). Phylogenetically, ibf. liinicoln and of the DNA is jO'!/o (as determined by HPLC). 130- strain SEBR 4S47T were also distinct (similarity of lated from oil-producing well. The type strain is 95%). a fzature which alone warrants to place SEBR SEBR 4S47T(= OCM -1.56). as a new species of the genus ,~Ieth~n7oplinzzís [2 I]. Strain SEBR 4WT was isolated from a subsurface Ac knowiedgments ecosystem. In contrast, Ji. liinicolsi was isolated from a small Italian swamp containing drilling waste and Funding in part from the Australian Research 56 B. 0llivic.r ri d./ FEMS Microbiology Leirers 147 (1997) 51-56

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