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Identification of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction
FEMS Microbiology Letters 180 (1999) 197^203
Identi¢cation of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction Rolf Daniel a , Falk Warnecke a , Joanna S. Potekhina b , Gerhard Gottschalk a
a;
*
Institut fu«r Mikrobiologie und Genetik der Georg-August-Universita«t, GrisebachstraMe 8, D-37077 Go«ttingen, Germany b Institute of Ecology of the Volga River Basin, Russian Academy of Sciences, 445003 Togliatti, Russia Received 2 September 1999; received in revised form 8 September 1999; accepted 8 September 1999
Abstract From enrichments with methanol and ferric pyrophosphate a coculture was isolated which coupled methanol oxidation to carbon dioxide with the reduction of Fe(III) to Fe(II). 16S rRNA gene analysis of the isolated syntrophic partners revealed 99.5% similarity to Clostridium sphenoides and 98.5% to Shewanella putrefaciens. Formation of Fe(II) coupled to methanol oxidation was confirmed by using strains of culture collections (C. sphenoides DSM 632 and S. putrefaciens DSM 9461). The importance of this process is discussed, also with respect to the anaerobic oxidation of methane. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Methanol oxidation; Dissimilatory Fe(III) reduction; Syntrophy; Methane oxidation ; Clostridium sphenoides ; Shewanella putrefaciens
1. Introduction Anaerobic oxidations coupled to the reduction of metals are important microbiological processes. Dissimilatory reduction of Fe(III) greatly a¡ects the geochemistry of non-sul¢dogenic anaerobic soils or sediments. A great variety of phylogenetically diverse microorganisms are able to conserve energy by coupling the oxidation of multicarbon organic acids and alcohols, H2 , aromatic compounds or the C1 substrate formate to the reduction of Fe(III) [1]. Among these are strict and facultative anaerobes, and meso-
* Corresponding author. Tel.: +49 (551) 393781; Fax: +49 (551) 393808; E-mail: [email protected]
philic as well as thermophilic organisms. Dissimilatory iron-reducing bacteria fall into two categories, those that completely oxidize multicarbon compounds to carbon dioxide and those that incompletely oxidize multicarbon organics to the level of acetate. The complete oxidizers include species from the Geobacter [2], Desulfuromonas [3], Desulfuromusa [4], and Geovibrio [5] genera, while the incomplete oxidizers include Shewanella [6] and Pelobacter species [7]. In connection with the general interest in the anaerobic oxidation of methane and di¤culties to experimentally prove such a process [8], we set up enrichments for the anaerobic oxidation of methanol coupled to Fe(III) reduction. To our knowledge no organism that uses methanol as substrate for dissimilatory iron reduction was identi¢ed so far. In this
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 8 6 - 3
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report we describe a syntrophic coculture that carries out this process.
2. Materials and methods 2.1. Bacterial strains Clostridium sphenoides DSM 632 and Shewanella putrefaciens DSM 9461 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). 2.2. Media and growth conditions Standard anaerobic techniques were used throughout the study [9]. The enrichment and the growth medium were boiled and cooled under a constant stream of 100% N2 or 80% N2 -20% CO2 , respectively. Both media were dispensed into aluminumsealed culture tubes under the same gas phase, capped with butyl rubber stoppers and sterilized by autoclaving. Additions to sterile media, inoculation, and sampling were done by using syringes and needles. The medium (A) for enrichment cultures contained the following (in g l31 ): ferric pyrophosphate, 10; NaH2 PO4 W2H2 O, 0.6; NH4 Cl, 1.5; MgSO4 W 7H2 O, 0.1; MgCl2 W6H2 O, 0.1; KCl, 0.1; yeast extract, 0.05; NaMoO4 W2H2 O, 0.001; vitamin solution according to Wolin et al. [10], 10 ml; and trace element solution SL6 [11], 3 ml; pH 7.0 to 7.5. Methanol (100 mM) was added as the electron donor. For preparation of agar plates 15 g l31 agar was added to the medium. The enrichment was initiated by adding 0.5 g (wet weight) of sediment to anaerobic £asks (125 ml) containing medium A (25 ml). The medium (B) used for growth and characterization of the isolated coculture contained the following (in grams per liter): ferric pyrophosphate, 11; NaH2 PO4 W2H2 O, 0.8; K2 HPO4 , 1.0; NH4 Cl, 1.5; MgSO4 W7H2 O, 0.1; MgCl2 W6H2 O, 0.1; KCl, 0.1; NaHCO3 , 4.0; NaMoO4 W2H2 O, 0.001; Na2 SeO3 , 0.001; vitamin solution according to Wolin et al. [10], 5 ml; and trace element solution SL6 [11], 3 ml; pH 7.0 to 7.2. The methanol concentration was varied from 25 to 5 mM. The use of di¡erent electron donors by strain FW1 was tested with enrichment medium without methanol. The electron donors were added
to the medium from sterile anaerobic stocks to give a ¢nal concentration of 10 to 30 mM. Ten ml of a 90% H2 -10% CO2 mixture or 90% CH4 -10% CO2 were added to the gas phase to test the use of H2 or CH4 as electron donors. C. sphenoides was grown on the medium described by Walther et al. [12]. S. putrefaciens was cultivated on LB medium [13]. 2.3. Analytical techniques Methanol and methane were determined gas chromatographically on a Poropack QS (80/100 mesh) column of 2 m length installed in a Packard Instruments Model 437 A (Chrompack, Frankfurt, Germany), which was equipped with a £ame ionization detector. The column temperature was 150³C, whereas the injector and detector temperature were 180³C. Gas chromatographic data were quanti¢ed with a Shimadzu (Kyoto, Japan) C-R2AX integrator. Fe(III) reduction was monitored by measuring the accumulation of Fe(II) over time. The amount of Fe(II) solubilized after 15 min in 0.5 N HCl was determined with ferrozine as described by Lovley and Philipps [14]. Cell growth was followed by determination of the protein content in the cultures with bovine serum albumin as the standard. A 0.6 ml sample was mixed with 0.5 ml double concentrated Bradford reagent [15], incubated for 20 min at room temperature, and then the A578 was measured. The production of 14 CO2 from [14 C]methanol (speci¢c radioactivity, 1 mCi mmol31 ) was monitored by liquid scintillation counting. A liquid scintillation counter model 1900 Tr (Packard Instruments, Frankfurt, Germany) was used. A 0.5 ml gas sample was removed directly from the gas phase and injected into an evacuated rubber-sealed glass tube containing a basic scintillation cocktail with 5 ml Perma£uor (Packard, Frankfurt, Germany) and 2 ml Carbosorb (Packard, Frankfurt, Germany) for trapping 14 CO2 . In order to trap volatile organic compounds in the gas phase, a 0.5 ml gas sample was injected in a tube containing only Perma£uor. For calculation of 14 CO2 formation the counts without Carbosorb were subtracted from those with Carbosorb. Identi¢cation of the isolated organisms, 16S rRNA gene sequencing and analysis of the fatty
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acid composition were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).
3. Results 3.1. Enrichment Enrichments were set up with sediments from various German rivers. The primary enrichment was performed in methanol-Fe(III) enrichment medium at 30³C. In the ¢rst two transfers the molybdate concentration of the medium was increased from 4 WM to 1 mM in order to inhibit sulfate reducers. CO2 in the atmosphere and bicarbonate in the medium were not rigorously excluded but not increased by addition to prevent acetogenesis from methanol and CO2 . The concentration of methanol was 100 mM. After seven transfers of six independent enrichments, further studies concentrated on a culture designated SB. This culture had been ob-
199
tained from enrichments with the sediment of a small river, named Aulebener Solbach. This river is situated near Nordhausen (Thuringia, Germany) and contains mixing water from deep aquifers and shallow ground water. The temperature is nearly constant throughout the year (about 16³C) and the content of chloride and sulfate ranges from 2 to 3 g per liter. Following transfer of SB into fresh medium methanol was completely decomposed within 13 days, Fe(III) pyrophosphate was reduced, but considerable amounts of methane were also produced. In subsequent transfers 20 mM bromoethanesulfonate was added to inhibit methanogenesis and the methanol concentration of the medium was decreased to 30 mM. After the 12th transfer of the SB enrichment culture Fe(III) reduction was observed as a change in the medium color from yellow to green, to a clearing of the medium and the formation of a white precipitate (probably vivianite [16]). Fe(III) pyrophosphate was completely reduced and approximately 15% of the initial methanol (30 mM) was degraded. Metha-
Table 1 Fatty acid composition of strain FW1, S. putrefaciens and S. alga Fatty acid
12:0 i13:0 13:0 i14:0 14:1g7c 14:0 i15:0 15:0 15:1g8c 15:1g6c i16:0 16:1g9c 16:1g7c 16:1g7t 16:0 i17:0 17:1g8c 17:1g6c 17:0 18:1g9c 18:1g7c other
% in: Strain FW1
S. putrefaciens
S. alga
2.5 5.9 1.6 0.9 not determined 2.1 19.2 7.6 1.5 1.4 not determined 2.1 20.6 not determined 8.6 0.9 12.4 not determined 2.1 1.7 1.7 7.2
0.1 to 2.6 1.0 to 3.6 0.0 to 0.5 6 0.1 to 0.3 0.0 to 6 0.1 1.5 to 5.9 8.8 to 24.2 1.4 to 7.5 0.0 to 0.7 0.0 to 0.3 0.0 to 6 0.1 0.0 to 2.4 25.2 to 31.4 1.0 to 1.7 27.9 to 31.0 6 0.1 to 1.6 2.7 to 3.7 0.0 to 0.4 0.6 to 1.6 1.1 to 3.6 2.4 to 3.9 0.6 to 6.2
0.1 0.5 0.1 0.8 6 0.1 0.7 17.8 4.0 6 0.1 6 0.1 1.1 not determined 16.3 0.2 13.3 0.5 14.7 1.4 4.6 6.0 7.2 5.6
Data for S. putrefaciens and S. alga were obtained by Bowman et al. [27]. The percentages for the fatty acid composition of S. alga do not add up to a 100%; an explanation for this is not given by Bowman et al. [27].
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Fig. 1. Reduction of Fe(III), CO2 production and growth by the coculture of FW1 and FW3 with methanol as electron donor. The growth experiment was performed in medium B. A: Autoclaved cells. B : Live cells.
nol decomposition was incomplete but no production of methane was recorded. Subsequently, selection on anaerobic methanol-Fe(III) pyrophosphate agar was started using 0.1 ml aliquots of 100 to 1036 dilutions of the SB enrichment culture. Various types of colonies grew within 2 to 14 days of incubation at 25³C. Fe(III) reduction was qualitatively
checked with a ferrozine solution. It appeared that it were the colorless colonies forming a white precipitate, which had produced Fe(II). Closer inspection revealed that these colonies were associated with small brownish colonies. Isolation of the two strains was successful, the white colony-forming strain (FW1) on Fe(III) pyrophosphate agar under an H2
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atmosphere, the brownish colony-forming strain (FW3) on a glucose-yeast extract agar. Neither strain alone grew on methanol plus Fe(III) but both strains together did, indicating that probably a syntrophic relationship existed. 3.2. Identi¢cation of the isolated strains FW1 and FW3 Strain FW1 is Gram-negative, facultatively anaerobic, non-spore-forming and motile by a polarly inserted £agellum. The cells are rods with a length of 1.5^3.5 Wm and a width of 0.5^0.7 Wm. The colonies formed on aerobic LB agar were slimy and opaque. Fe(III) reduction occurred with a variety of electron donors including H2 /CO2 , formate, pyruvate, lactate, glucose or yeast extract. No reduction was observed with methanol or acetate as substrates. 16S rRNA gene analysis identi¢ed strain FW1 as a member of the genus Shewanella. FW1 shared 98.5% similarity with S. putrefaciens and Shewanella alga. The fatty acid composition of FW1 is shown in Table 1. The pattern of FW1 is similar to S. putrefaciens with exceptions for palmitic acid (16:0) and unsaturated heptadecanoic acid (17:1g8c) where the similarity is greater to S. alga. We conclude especially on the basis of the fatty acid composition that FW1 represents a new strain within the species S. putrefaciens. Strain FW3 is Gram-positive, strictly anaerobic, and spore-forming. The strain grew axenically in the enrichment medium with glucose as substrate and without Fe(III) reduction. FW3 did not grow with methanol in axenic culture. 16S rRNA gene analysis revealed 99.5% similarity to C. sphenoides. In summary, two well-characterized species carried out anaerobic methanol oxidation coupled to Fe(III) reduction. Subsequently, it was possible to con¢rm this using strains from culture collections. This was done with C. sphenoides DSM 632 and S. putrefaciens DSM 9461. 3.3. Synthrophic coculture of strains FW1 and FW3 The anaerobic coculture was initiated by transferring an inoculum of formate-grown FW1 and glucose-grown FW3 into fresh methanol-Fe(III) enrichment medium. The initial methanol concentration was 25 mM. The coculture degraded 3 to 5 mM methanol, and the available Fe(III) (approximately
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15 to 20 mM) was completely reduced. Pure cultures of FW1, S. putrefaciens, FW3 or C. sphenoides did not degrade methanol. In addition growth or iron reduction was not observed. After several consecutive transfers of the coculture growth and iron reduction became unreproducible. Subsequently, the medium was optimized. Addition of bicarbonate resulted in reproducible growth of the coculture. Therefore medium B (see Section 2) was employed for further experiments. FW1 and FW3 grew axenically in medium B with formate or glucose as substrate, respectively. Both strains did not grow with methanol in axenic culture. Similar results were obtained for the corresponding type strains. The coculture of FW1 and FW3 was able to reduce Fe(III) coupled to methanol oxidation and growth in medium B. Production of approximately 17 mM of Fe(II) was associated with the disappearance of 3 to 4 mM methanol. Other organic acids, such as acetate, were not detected in the medium. Since the changes in methanol concentration are small and determination of the methanol concentration by gas chromatography is not very sensitive in this range, radioactive methanol was used to show the production of CO2 from methanol by the coculture. Oxidation of 14 C-labeled methanol to 14 CO2 occurred concomitantly with growth and the reduction of Fe(III) by the coculture (Fig. 1); oxidation of [14 C]methanol by autoclaved cells (Fig. 1), by axenic cultures or in the absence of Fe(III) was not detectable or minimal. Formation of 14 CO2 from [14 C]methanol was also recorded by using a coculture of C. sphenoides DSM 632 and S. putrefaciens DSM 9461. Anaerobic oxidation of methane coupled to Fe(III) reduction by the coculture was not observed.
4. Discussion Dissimilatory Fe(III) reducers exhibit a rather broad substrate spectrum. It ranges from H2 +CO2 , formate and acetate to compounds such as toluene or phenol [1]. So it was unexpected that methanol is utilized by a syntrophic coculture. Since CO2 is the ¢nal product of methanol oxidation, and H2 and perhaps formate is used for Fe(III) reduction, the process can be described by the following equations:
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C. sphenoides CH3 OH 2H2 O !
HCO3 3
3H2 H
1
or CH3 OH H2 O ! HCOO3 2H2 H
2
S. putrefaciens 3H2 6Fe3 ! 6Fe2 6H
3
or 2H2 HCOO3 6Fe3 H2 O ! 6Fe2 HCO3 3 6H
4
Secretion of formate by C. sphenoides and its utilization by S. putrefaciens cannot be ruled out. Since the overall process is rather slow and takes 2 weeks under the conditions employed (see Fig. 1), facilitated di¡usion processes of formate not requiring metabolic energy and not charging the overall energy balance may account for the coupling between C. sphenoides and S. putrefaciens (equations (2) and (4)). Formate transfer was demonstrated for a coculture of Eubacterium acidaminophilum and Desulfovibrio baarsii during growth on alanine or aspartate and sulfate [17]. Acetate is unlikely to play a major role as mediator, because it is not utilized by S. putrefaciens, even when incubated for 4 weeks. Although Fe(III) reducers have been shown to decrease the partial pressure of H2 below values reported for methanogenic and sul¢dogenic microorganisms, they have not often been employed in syntrophic systems. Knight et al. [18] described a facultative syntrophic coculture of Aeromonas veronii and S. alga on citrate. Whereas S. putrefaciens is known as H2 utilizer, the ability of C. sphenoides to oxidize methanol under these conditions was a surprising result. This clostridium is saccharolytic, and ferments citrate [12,19]. Under phosphate limitation it forms 1,2-propanediol via the methyl-glyoxal bypass [20]. So it exhibits some metabolic £exibility which is documented further by the ¢ndings reported here. Which pathway is used for methanol oxidation is unknown and must be investigated. The question remains whether the ¢ndings reported here can serve as a working hypothesis to
unravel anaerobic methane oxidation. Such a process has been proposed on the basis of carbon isotope discrimination experiments [21]. Anaerobic methane oxidation has been attributed to the activity of methanogenic microorganisms [22] or sulfate-reducing bacteria [8]. The free energy change of methane oxidation coupled to sulfate re0 duction according to equation (5) is vG0 = 318 kJ reaction31 . 3 3 CH4 SO23 4 ! HCO3 HS H2 O
5
It is below the value of 320 kJ reaction31 , which is considered to be the smallest quantum of metabolically convertible energy. This amount of energy is by far insu¤cient to sustain a coculture [23]. In correspondence with this all attempts to couple methane oxidation with sulfate reduction were unsuccessful. There is still a large di¡erence between methanol and methane oxidation, both mechanistically and thermodynamically. Methanol oxidation according to equation (1) is associated with a free energy 0 change of vG0 = +23.5 kJ reaction31 [24], methane oxidation to H2 and bicarbonate (equation (6)) with 0 a vG0 = +131 kJ reaction31 [23]: CH4 3H2 O ! HCO3 3 4H2 H
6
However, because of the exergonic nature of Fe3 0 reduction (Fe3 +1/2H2 CFe2 +H , vG0 = 3114.2 kJ reaction31 ) the overall process is feasible: 2 CH4 8Fe3 3H2 O ! HCO3 9H 3 8Fe
7 0
vG0 = 3782 kJ reaction31 . This highly negative value does not take into account the low solubility of Fe3 at neutral pH. A syntrophic process will be slow because it has to operate at extremely low concentrations of H2 (less than 1 nM). Otherwise equation (6) will not proceed in the direction of H2 formation. Which organism could be the methane oxidizer? Besides strict anaerobes such as reversible methanogens [23,25] methanotrophic bacteria have to be considered. A positive e¡ect of CH4 plus Fe(III) on the survival of methanotrophic bacteria under strictly anaerobic conditions was observed [26]. On the basis of the results reported here, these experiments have
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to be taken up again, but in the presence of a Fe(III) reducer such as S. putrefaciens.
Acknowledgements This work was supported by a fellowship of the Deutsche Forschungsgemeinschaft, Bonn to one of us (J.S.P.) and by funds of the Akademie der Wissenschaften zu Go«ttingen.
References [1] Lovley, D.R. (1997) Microbial Fe(III)-reduction in subsurface environments. FEMS Microbiol. Rev. 20, 305^313. [2] Caccavo, F., Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F. and McInerney, M.J. (1994) Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metalreducing microorganism. Appl. Environ. Microbiol. 60, 3752^ 3759. [3] Coates, J.D., Lonergan, D.J. and Lovley, D.R. (1995) Desulfuromonas palmitatis sp. nov., a marine dissimilatory Fe(III) reducer that can oxidize long-chain fatty acids. Arch. Microbiol. 164, 406^413. [4] Lonergan, D.J., Jenter, H., Coates, J.D., Schmidt, T. and Lovley, D.R. (1996) Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. J. Bacteriol. 178, 2402^2408. [5] Caccavo, F., Coates, J.D., Rosello-Mora, R.A., Ludwig, W., Schleifer, K.H., Lovley, D.R. and McInerney, M.J. (1996) Geovibrio ferrireducens, a phylogenetic distinct Fe(III)-reducing bacterium. Arch. Microbiol. 165, 370^376. [6] Lovley, D.R., Phillips, E.J.P. and Lonergan, D.J. (1989) Hydrogen and formate oxidation coupled to dissimilatory reduction of iron or manganese by Alteromonas putrefaciens. Appl. Environ. Microbiol. 55, 700^706. [7] Lovley, D.R., Phillips, E.J.P., Lonergan, D.J. and Widman, P.K. (1995) Fe(III) and S0 reduction by Pelobacter carbinolicus. Appl. Environ. Microbiol. 61, 2132^2138. [8] Hoehler, T.M., Alperin, M.J., Albert, D.B. and Martens, C.S. (1994) Field and laboratory studies of methane oxidation in an anoxic marine sediment : Evidence for a methanogen sulfate-reducer consortium. Glob. Biogeochem. Cycles 8, 451^ 463. [9] Hungate, R.E. (1969) A role tube method for cultivation of strict anaerobes. In: Methods in Microbiology (Norris, J.R. and Ribbons, D.W., Eds.), Vol. 3, pp. 117^132. Academic Press, New York. [10] Wolin, E.A., Wolfe, R.S. and Wolin, M.J. (1964) Viologen dye inhibition of methane formation by Methanobacterium omelianskii. J. Bacteriol. 87, 993^998. ë ber das Vitamin B12[11] Pfennig, N. and Lippert, K.D. (1966) U Bedu«rfnis phototropher Schwefelbakterien. Arch. Microbiol. 55, 245^256.
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[12] Walther, R., Hippe, H. and Gottschalk, G. (1977) Citrate, a speci¢c substrate for the isolation of Clostridium sphenoides. Appl. Environ. Microbiol. 33, 955^962. [13] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [14] Lovley, D.R. and Phililips, E.J.P. (1986) Availability of ferric iron for microbial reduction in bottom sediments of the freshwater tidal Potomac River. Appl. Environ. Microbiol. 52, 751^757. [15] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [16] Lovley, D.R. (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55, 259^287. [17] Zindel, U., Freudenberg, W., Rieth, M., Andreesen, J.R., Schnell, J. and Widdel, F. (1988) Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate. Description and enzymatic studies. Arch. Microbiol. 150, 254^266. [18] Knight, V., Caccavo, F., Wudyka, S. and Blakemore, R. (1996) Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii. Arch. Microbiol. 166, 269^274. [19] Antranikian, G., Friese, C., Quentmeier, A., Hippe, H. and Gottschalk, G. (1984) Distribution of the ability for citrate utilization amongst clostridia. Arch. Microbiol. 138, 179^182. [20] Tran-Dinh, K. and Gottschalk, G. (1985) Formation of D(-)1, 2-propanediol and D(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation. Arch. Microbiol. 142, 87^92. [21] Iversen, N. (1996) Methane oxidation in coastal marine environments. In: Microbiology of Atmospheric Trace Gases. Sources, Sinks and Global Change Processes (Murrell, J.C. and Kelly, D.P., Eds.), pp. 51^68. Springer Verlag, Berlin. [22] Zehnder, A.J.B. and Brock, T.D. (1979) Methane formation and methane oxidation by methanogenic bacteria. J. Bacteriol. 137, 420^432. [23] Schink, B. (1997) Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61, 262^280. [24] Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bact. Rev. 41, 100^180. [25] Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G. and DeLong, E.F. (1999) Methane-consuming archaebacteria in marine sediments. Nature 398, 802^805. [26] Potekhina, J.S., Umanskaja, M.V. and Vasneva, J.P. (1990) Reduction of ferric iron compounds by Methylomonas methanica. Proc. USSR Acad. Sci. 315, 731^732. [27] Bowman, J.P., McCammon, S.A., Nichols, D.S., Skerratt, J.H., Rea, S.M., Nichols, P.D. and McMeekin, T.A. (1997) Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20 g3) and grow anaerobically by dissimilatory Fe(III) reduction. Int. J. Syst. Bacteriol. 47, 1040^ 1047.
FEMSLE 9067 28-10-99
Identi¢cation of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction Rolf Daniel a , Falk Warnecke a , Joanna S. Potekhina b , Gerhard Gottschalk a
a;
*
Institut fu«r Mikrobiologie und Genetik der Georg-August-Universita«t, GrisebachstraMe 8, D-37077 Go«ttingen, Germany b Institute of Ecology of the Volga River Basin, Russian Academy of Sciences, 445003 Togliatti, Russia Received 2 September 1999; received in revised form 8 September 1999; accepted 8 September 1999
Abstract From enrichments with methanol and ferric pyrophosphate a coculture was isolated which coupled methanol oxidation to carbon dioxide with the reduction of Fe(III) to Fe(II). 16S rRNA gene analysis of the isolated syntrophic partners revealed 99.5% similarity to Clostridium sphenoides and 98.5% to Shewanella putrefaciens. Formation of Fe(II) coupled to methanol oxidation was confirmed by using strains of culture collections (C. sphenoides DSM 632 and S. putrefaciens DSM 9461). The importance of this process is discussed, also with respect to the anaerobic oxidation of methane. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Methanol oxidation; Dissimilatory Fe(III) reduction; Syntrophy; Methane oxidation ; Clostridium sphenoides ; Shewanella putrefaciens
1. Introduction Anaerobic oxidations coupled to the reduction of metals are important microbiological processes. Dissimilatory reduction of Fe(III) greatly a¡ects the geochemistry of non-sul¢dogenic anaerobic soils or sediments. A great variety of phylogenetically diverse microorganisms are able to conserve energy by coupling the oxidation of multicarbon organic acids and alcohols, H2 , aromatic compounds or the C1 substrate formate to the reduction of Fe(III) [1]. Among these are strict and facultative anaerobes, and meso-
* Corresponding author. Tel.: +49 (551) 393781; Fax: +49 (551) 393808; E-mail: [email protected]
philic as well as thermophilic organisms. Dissimilatory iron-reducing bacteria fall into two categories, those that completely oxidize multicarbon compounds to carbon dioxide and those that incompletely oxidize multicarbon organics to the level of acetate. The complete oxidizers include species from the Geobacter [2], Desulfuromonas [3], Desulfuromusa [4], and Geovibrio [5] genera, while the incomplete oxidizers include Shewanella [6] and Pelobacter species [7]. In connection with the general interest in the anaerobic oxidation of methane and di¤culties to experimentally prove such a process [8], we set up enrichments for the anaerobic oxidation of methanol coupled to Fe(III) reduction. To our knowledge no organism that uses methanol as substrate for dissimilatory iron reduction was identi¢ed so far. In this
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 8 6 - 3
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report we describe a syntrophic coculture that carries out this process.
2. Materials and methods 2.1. Bacterial strains Clostridium sphenoides DSM 632 and Shewanella putrefaciens DSM 9461 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). 2.2. Media and growth conditions Standard anaerobic techniques were used throughout the study [9]. The enrichment and the growth medium were boiled and cooled under a constant stream of 100% N2 or 80% N2 -20% CO2 , respectively. Both media were dispensed into aluminumsealed culture tubes under the same gas phase, capped with butyl rubber stoppers and sterilized by autoclaving. Additions to sterile media, inoculation, and sampling were done by using syringes and needles. The medium (A) for enrichment cultures contained the following (in g l31 ): ferric pyrophosphate, 10; NaH2 PO4 W2H2 O, 0.6; NH4 Cl, 1.5; MgSO4 W 7H2 O, 0.1; MgCl2 W6H2 O, 0.1; KCl, 0.1; yeast extract, 0.05; NaMoO4 W2H2 O, 0.001; vitamin solution according to Wolin et al. [10], 10 ml; and trace element solution SL6 [11], 3 ml; pH 7.0 to 7.5. Methanol (100 mM) was added as the electron donor. For preparation of agar plates 15 g l31 agar was added to the medium. The enrichment was initiated by adding 0.5 g (wet weight) of sediment to anaerobic £asks (125 ml) containing medium A (25 ml). The medium (B) used for growth and characterization of the isolated coculture contained the following (in grams per liter): ferric pyrophosphate, 11; NaH2 PO4 W2H2 O, 0.8; K2 HPO4 , 1.0; NH4 Cl, 1.5; MgSO4 W7H2 O, 0.1; MgCl2 W6H2 O, 0.1; KCl, 0.1; NaHCO3 , 4.0; NaMoO4 W2H2 O, 0.001; Na2 SeO3 , 0.001; vitamin solution according to Wolin et al. [10], 5 ml; and trace element solution SL6 [11], 3 ml; pH 7.0 to 7.2. The methanol concentration was varied from 25 to 5 mM. The use of di¡erent electron donors by strain FW1 was tested with enrichment medium without methanol. The electron donors were added
to the medium from sterile anaerobic stocks to give a ¢nal concentration of 10 to 30 mM. Ten ml of a 90% H2 -10% CO2 mixture or 90% CH4 -10% CO2 were added to the gas phase to test the use of H2 or CH4 as electron donors. C. sphenoides was grown on the medium described by Walther et al. [12]. S. putrefaciens was cultivated on LB medium [13]. 2.3. Analytical techniques Methanol and methane were determined gas chromatographically on a Poropack QS (80/100 mesh) column of 2 m length installed in a Packard Instruments Model 437 A (Chrompack, Frankfurt, Germany), which was equipped with a £ame ionization detector. The column temperature was 150³C, whereas the injector and detector temperature were 180³C. Gas chromatographic data were quanti¢ed with a Shimadzu (Kyoto, Japan) C-R2AX integrator. Fe(III) reduction was monitored by measuring the accumulation of Fe(II) over time. The amount of Fe(II) solubilized after 15 min in 0.5 N HCl was determined with ferrozine as described by Lovley and Philipps [14]. Cell growth was followed by determination of the protein content in the cultures with bovine serum albumin as the standard. A 0.6 ml sample was mixed with 0.5 ml double concentrated Bradford reagent [15], incubated for 20 min at room temperature, and then the A578 was measured. The production of 14 CO2 from [14 C]methanol (speci¢c radioactivity, 1 mCi mmol31 ) was monitored by liquid scintillation counting. A liquid scintillation counter model 1900 Tr (Packard Instruments, Frankfurt, Germany) was used. A 0.5 ml gas sample was removed directly from the gas phase and injected into an evacuated rubber-sealed glass tube containing a basic scintillation cocktail with 5 ml Perma£uor (Packard, Frankfurt, Germany) and 2 ml Carbosorb (Packard, Frankfurt, Germany) for trapping 14 CO2 . In order to trap volatile organic compounds in the gas phase, a 0.5 ml gas sample was injected in a tube containing only Perma£uor. For calculation of 14 CO2 formation the counts without Carbosorb were subtracted from those with Carbosorb. Identi¢cation of the isolated organisms, 16S rRNA gene sequencing and analysis of the fatty
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acid composition were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).
3. Results 3.1. Enrichment Enrichments were set up with sediments from various German rivers. The primary enrichment was performed in methanol-Fe(III) enrichment medium at 30³C. In the ¢rst two transfers the molybdate concentration of the medium was increased from 4 WM to 1 mM in order to inhibit sulfate reducers. CO2 in the atmosphere and bicarbonate in the medium were not rigorously excluded but not increased by addition to prevent acetogenesis from methanol and CO2 . The concentration of methanol was 100 mM. After seven transfers of six independent enrichments, further studies concentrated on a culture designated SB. This culture had been ob-
199
tained from enrichments with the sediment of a small river, named Aulebener Solbach. This river is situated near Nordhausen (Thuringia, Germany) and contains mixing water from deep aquifers and shallow ground water. The temperature is nearly constant throughout the year (about 16³C) and the content of chloride and sulfate ranges from 2 to 3 g per liter. Following transfer of SB into fresh medium methanol was completely decomposed within 13 days, Fe(III) pyrophosphate was reduced, but considerable amounts of methane were also produced. In subsequent transfers 20 mM bromoethanesulfonate was added to inhibit methanogenesis and the methanol concentration of the medium was decreased to 30 mM. After the 12th transfer of the SB enrichment culture Fe(III) reduction was observed as a change in the medium color from yellow to green, to a clearing of the medium and the formation of a white precipitate (probably vivianite [16]). Fe(III) pyrophosphate was completely reduced and approximately 15% of the initial methanol (30 mM) was degraded. Metha-
Table 1 Fatty acid composition of strain FW1, S. putrefaciens and S. alga Fatty acid
12:0 i13:0 13:0 i14:0 14:1g7c 14:0 i15:0 15:0 15:1g8c 15:1g6c i16:0 16:1g9c 16:1g7c 16:1g7t 16:0 i17:0 17:1g8c 17:1g6c 17:0 18:1g9c 18:1g7c other
% in: Strain FW1
S. putrefaciens
S. alga
2.5 5.9 1.6 0.9 not determined 2.1 19.2 7.6 1.5 1.4 not determined 2.1 20.6 not determined 8.6 0.9 12.4 not determined 2.1 1.7 1.7 7.2
0.1 to 2.6 1.0 to 3.6 0.0 to 0.5 6 0.1 to 0.3 0.0 to 6 0.1 1.5 to 5.9 8.8 to 24.2 1.4 to 7.5 0.0 to 0.7 0.0 to 0.3 0.0 to 6 0.1 0.0 to 2.4 25.2 to 31.4 1.0 to 1.7 27.9 to 31.0 6 0.1 to 1.6 2.7 to 3.7 0.0 to 0.4 0.6 to 1.6 1.1 to 3.6 2.4 to 3.9 0.6 to 6.2
0.1 0.5 0.1 0.8 6 0.1 0.7 17.8 4.0 6 0.1 6 0.1 1.1 not determined 16.3 0.2 13.3 0.5 14.7 1.4 4.6 6.0 7.2 5.6
Data for S. putrefaciens and S. alga were obtained by Bowman et al. [27]. The percentages for the fatty acid composition of S. alga do not add up to a 100%; an explanation for this is not given by Bowman et al. [27].
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Fig. 1. Reduction of Fe(III), CO2 production and growth by the coculture of FW1 and FW3 with methanol as electron donor. The growth experiment was performed in medium B. A: Autoclaved cells. B : Live cells.
nol decomposition was incomplete but no production of methane was recorded. Subsequently, selection on anaerobic methanol-Fe(III) pyrophosphate agar was started using 0.1 ml aliquots of 100 to 1036 dilutions of the SB enrichment culture. Various types of colonies grew within 2 to 14 days of incubation at 25³C. Fe(III) reduction was qualitatively
checked with a ferrozine solution. It appeared that it were the colorless colonies forming a white precipitate, which had produced Fe(II). Closer inspection revealed that these colonies were associated with small brownish colonies. Isolation of the two strains was successful, the white colony-forming strain (FW1) on Fe(III) pyrophosphate agar under an H2
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atmosphere, the brownish colony-forming strain (FW3) on a glucose-yeast extract agar. Neither strain alone grew on methanol plus Fe(III) but both strains together did, indicating that probably a syntrophic relationship existed. 3.2. Identi¢cation of the isolated strains FW1 and FW3 Strain FW1 is Gram-negative, facultatively anaerobic, non-spore-forming and motile by a polarly inserted £agellum. The cells are rods with a length of 1.5^3.5 Wm and a width of 0.5^0.7 Wm. The colonies formed on aerobic LB agar were slimy and opaque. Fe(III) reduction occurred with a variety of electron donors including H2 /CO2 , formate, pyruvate, lactate, glucose or yeast extract. No reduction was observed with methanol or acetate as substrates. 16S rRNA gene analysis identi¢ed strain FW1 as a member of the genus Shewanella. FW1 shared 98.5% similarity with S. putrefaciens and Shewanella alga. The fatty acid composition of FW1 is shown in Table 1. The pattern of FW1 is similar to S. putrefaciens with exceptions for palmitic acid (16:0) and unsaturated heptadecanoic acid (17:1g8c) where the similarity is greater to S. alga. We conclude especially on the basis of the fatty acid composition that FW1 represents a new strain within the species S. putrefaciens. Strain FW3 is Gram-positive, strictly anaerobic, and spore-forming. The strain grew axenically in the enrichment medium with glucose as substrate and without Fe(III) reduction. FW3 did not grow with methanol in axenic culture. 16S rRNA gene analysis revealed 99.5% similarity to C. sphenoides. In summary, two well-characterized species carried out anaerobic methanol oxidation coupled to Fe(III) reduction. Subsequently, it was possible to con¢rm this using strains from culture collections. This was done with C. sphenoides DSM 632 and S. putrefaciens DSM 9461. 3.3. Synthrophic coculture of strains FW1 and FW3 The anaerobic coculture was initiated by transferring an inoculum of formate-grown FW1 and glucose-grown FW3 into fresh methanol-Fe(III) enrichment medium. The initial methanol concentration was 25 mM. The coculture degraded 3 to 5 mM methanol, and the available Fe(III) (approximately
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15 to 20 mM) was completely reduced. Pure cultures of FW1, S. putrefaciens, FW3 or C. sphenoides did not degrade methanol. In addition growth or iron reduction was not observed. After several consecutive transfers of the coculture growth and iron reduction became unreproducible. Subsequently, the medium was optimized. Addition of bicarbonate resulted in reproducible growth of the coculture. Therefore medium B (see Section 2) was employed for further experiments. FW1 and FW3 grew axenically in medium B with formate or glucose as substrate, respectively. Both strains did not grow with methanol in axenic culture. Similar results were obtained for the corresponding type strains. The coculture of FW1 and FW3 was able to reduce Fe(III) coupled to methanol oxidation and growth in medium B. Production of approximately 17 mM of Fe(II) was associated with the disappearance of 3 to 4 mM methanol. Other organic acids, such as acetate, were not detected in the medium. Since the changes in methanol concentration are small and determination of the methanol concentration by gas chromatography is not very sensitive in this range, radioactive methanol was used to show the production of CO2 from methanol by the coculture. Oxidation of 14 C-labeled methanol to 14 CO2 occurred concomitantly with growth and the reduction of Fe(III) by the coculture (Fig. 1); oxidation of [14 C]methanol by autoclaved cells (Fig. 1), by axenic cultures or in the absence of Fe(III) was not detectable or minimal. Formation of 14 CO2 from [14 C]methanol was also recorded by using a coculture of C. sphenoides DSM 632 and S. putrefaciens DSM 9461. Anaerobic oxidation of methane coupled to Fe(III) reduction by the coculture was not observed.
4. Discussion Dissimilatory Fe(III) reducers exhibit a rather broad substrate spectrum. It ranges from H2 +CO2 , formate and acetate to compounds such as toluene or phenol [1]. So it was unexpected that methanol is utilized by a syntrophic coculture. Since CO2 is the ¢nal product of methanol oxidation, and H2 and perhaps formate is used for Fe(III) reduction, the process can be described by the following equations:
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C. sphenoides CH3 OH 2H2 O !
HCO3 3
3H2 H
1
or CH3 OH H2 O ! HCOO3 2H2 H
2
S. putrefaciens 3H2 6Fe3 ! 6Fe2 6H
3
or 2H2 HCOO3 6Fe3 H2 O ! 6Fe2 HCO3 3 6H
4
Secretion of formate by C. sphenoides and its utilization by S. putrefaciens cannot be ruled out. Since the overall process is rather slow and takes 2 weeks under the conditions employed (see Fig. 1), facilitated di¡usion processes of formate not requiring metabolic energy and not charging the overall energy balance may account for the coupling between C. sphenoides and S. putrefaciens (equations (2) and (4)). Formate transfer was demonstrated for a coculture of Eubacterium acidaminophilum and Desulfovibrio baarsii during growth on alanine or aspartate and sulfate [17]. Acetate is unlikely to play a major role as mediator, because it is not utilized by S. putrefaciens, even when incubated for 4 weeks. Although Fe(III) reducers have been shown to decrease the partial pressure of H2 below values reported for methanogenic and sul¢dogenic microorganisms, they have not often been employed in syntrophic systems. Knight et al. [18] described a facultative syntrophic coculture of Aeromonas veronii and S. alga on citrate. Whereas S. putrefaciens is known as H2 utilizer, the ability of C. sphenoides to oxidize methanol under these conditions was a surprising result. This clostridium is saccharolytic, and ferments citrate [12,19]. Under phosphate limitation it forms 1,2-propanediol via the methyl-glyoxal bypass [20]. So it exhibits some metabolic £exibility which is documented further by the ¢ndings reported here. Which pathway is used for methanol oxidation is unknown and must be investigated. The question remains whether the ¢ndings reported here can serve as a working hypothesis to
unravel anaerobic methane oxidation. Such a process has been proposed on the basis of carbon isotope discrimination experiments [21]. Anaerobic methane oxidation has been attributed to the activity of methanogenic microorganisms [22] or sulfate-reducing bacteria [8]. The free energy change of methane oxidation coupled to sulfate re0 duction according to equation (5) is vG0 = 318 kJ reaction31 . 3 3 CH4 SO23 4 ! HCO3 HS H2 O
5
It is below the value of 320 kJ reaction31 , which is considered to be the smallest quantum of metabolically convertible energy. This amount of energy is by far insu¤cient to sustain a coculture [23]. In correspondence with this all attempts to couple methane oxidation with sulfate reduction were unsuccessful. There is still a large di¡erence between methanol and methane oxidation, both mechanistically and thermodynamically. Methanol oxidation according to equation (1) is associated with a free energy 0 change of vG0 = +23.5 kJ reaction31 [24], methane oxidation to H2 and bicarbonate (equation (6)) with 0 a vG0 = +131 kJ reaction31 [23]: CH4 3H2 O ! HCO3 3 4H2 H
6
However, because of the exergonic nature of Fe3 0 reduction (Fe3 +1/2H2 CFe2 +H , vG0 = 3114.2 kJ reaction31 ) the overall process is feasible: 2 CH4 8Fe3 3H2 O ! HCO3 9H 3 8Fe
7 0
vG0 = 3782 kJ reaction31 . This highly negative value does not take into account the low solubility of Fe3 at neutral pH. A syntrophic process will be slow because it has to operate at extremely low concentrations of H2 (less than 1 nM). Otherwise equation (6) will not proceed in the direction of H2 formation. Which organism could be the methane oxidizer? Besides strict anaerobes such as reversible methanogens [23,25] methanotrophic bacteria have to be considered. A positive e¡ect of CH4 plus Fe(III) on the survival of methanotrophic bacteria under strictly anaerobic conditions was observed [26]. On the basis of the results reported here, these experiments have
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to be taken up again, but in the presence of a Fe(III) reducer such as S. putrefaciens.
Acknowledgements This work was supported by a fellowship of the Deutsche Forschungsgemeinschaft, Bonn to one of us (J.S.P.) and by funds of the Akademie der Wissenschaften zu Go«ttingen.
References [1] Lovley, D.R. (1997) Microbial Fe(III)-reduction in subsurface environments. FEMS Microbiol. Rev. 20, 305^313. [2] Caccavo, F., Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F. and McInerney, M.J. (1994) Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metalreducing microorganism. Appl. Environ. Microbiol. 60, 3752^ 3759. [3] Coates, J.D., Lonergan, D.J. and Lovley, D.R. (1995) Desulfuromonas palmitatis sp. nov., a marine dissimilatory Fe(III) reducer that can oxidize long-chain fatty acids. Arch. Microbiol. 164, 406^413. [4] Lonergan, D.J., Jenter, H., Coates, J.D., Schmidt, T. and Lovley, D.R. (1996) Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. J. Bacteriol. 178, 2402^2408. [5] Caccavo, F., Coates, J.D., Rosello-Mora, R.A., Ludwig, W., Schleifer, K.H., Lovley, D.R. and McInerney, M.J. (1996) Geovibrio ferrireducens, a phylogenetic distinct Fe(III)-reducing bacterium. Arch. Microbiol. 165, 370^376. [6] Lovley, D.R., Phillips, E.J.P. and Lonergan, D.J. (1989) Hydrogen and formate oxidation coupled to dissimilatory reduction of iron or manganese by Alteromonas putrefaciens. Appl. Environ. Microbiol. 55, 700^706. [7] Lovley, D.R., Phillips, E.J.P., Lonergan, D.J. and Widman, P.K. (1995) Fe(III) and S0 reduction by Pelobacter carbinolicus. Appl. Environ. Microbiol. 61, 2132^2138. [8] Hoehler, T.M., Alperin, M.J., Albert, D.B. and Martens, C.S. (1994) Field and laboratory studies of methane oxidation in an anoxic marine sediment : Evidence for a methanogen sulfate-reducer consortium. Glob. Biogeochem. Cycles 8, 451^ 463. [9] Hungate, R.E. (1969) A role tube method for cultivation of strict anaerobes. In: Methods in Microbiology (Norris, J.R. and Ribbons, D.W., Eds.), Vol. 3, pp. 117^132. Academic Press, New York. [10] Wolin, E.A., Wolfe, R.S. and Wolin, M.J. (1964) Viologen dye inhibition of methane formation by Methanobacterium omelianskii. J. Bacteriol. 87, 993^998. ë ber das Vitamin B12[11] Pfennig, N. and Lippert, K.D. (1966) U Bedu«rfnis phototropher Schwefelbakterien. Arch. Microbiol. 55, 245^256.
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[12] Walther, R., Hippe, H. and Gottschalk, G. (1977) Citrate, a speci¢c substrate for the isolation of Clostridium sphenoides. Appl. Environ. Microbiol. 33, 955^962. [13] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [14] Lovley, D.R. and Phililips, E.J.P. (1986) Availability of ferric iron for microbial reduction in bottom sediments of the freshwater tidal Potomac River. Appl. Environ. Microbiol. 52, 751^757. [15] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [16] Lovley, D.R. (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55, 259^287. [17] Zindel, U., Freudenberg, W., Rieth, M., Andreesen, J.R., Schnell, J. and Widdel, F. (1988) Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate. Description and enzymatic studies. Arch. Microbiol. 150, 254^266. [18] Knight, V., Caccavo, F., Wudyka, S. and Blakemore, R. (1996) Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii. Arch. Microbiol. 166, 269^274. [19] Antranikian, G., Friese, C., Quentmeier, A., Hippe, H. and Gottschalk, G. (1984) Distribution of the ability for citrate utilization amongst clostridia. Arch. Microbiol. 138, 179^182. [20] Tran-Dinh, K. and Gottschalk, G. (1985) Formation of D(-)1, 2-propanediol and D(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation. Arch. Microbiol. 142, 87^92. [21] Iversen, N. (1996) Methane oxidation in coastal marine environments. In: Microbiology of Atmospheric Trace Gases. Sources, Sinks and Global Change Processes (Murrell, J.C. and Kelly, D.P., Eds.), pp. 51^68. Springer Verlag, Berlin. [22] Zehnder, A.J.B. and Brock, T.D. (1979) Methane formation and methane oxidation by methanogenic bacteria. J. Bacteriol. 137, 420^432. [23] Schink, B. (1997) Energetics of syntrophic cooperation in methanogenic degradation. Microbiol. Mol. Biol. Rev. 61, 262^280. [24] Thauer, R.K., Jungermann, K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bact. Rev. 41, 100^180. [25] Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G. and DeLong, E.F. (1999) Methane-consuming archaebacteria in marine sediments. Nature 398, 802^805. [26] Potekhina, J.S., Umanskaja, M.V. and Vasneva, J.P. (1990) Reduction of ferric iron compounds by Methylomonas methanica. Proc. USSR Acad. Sci. 315, 731^732. [27] Bowman, J.P., McCammon, S.A., Nichols, D.S., Skerratt, J.H., Rea, S.M., Nichols, P.D. and McMeekin, T.A. (1997) Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20 g3) and grow anaerobically by dissimilatory Fe(III) reduction. Int. J. Syst. Bacteriol. 47, 1040^ 1047.
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